RESUMEN
In Arabidopsis thaliana, two genes encode the E2 subunit of the 2-oxoglutarate dehydrogenase (2-OGDH), a multimeric complex composed of three subunits. To functionally characterize the isoforms of E2 subunit, we isolated Arabidopsis mutant lines for each gene encoding the E2 subunit and performed a detailed molecular and physiological characterization of the plants under controlled growth conditions. The functional lack of expression of E2 subunit isoforms of 2-OGDH increased plant growth, reduced dark respiration and altered carbohydrate metabolism without changes in the photosynthetic rate. Interestingly, plants from e2-ogdh lines also exhibited reduced seed weight without alterations in total seed number. We additionally observed that downregulation of 2-OGDH activity led to minor changes in the levels of tricarboxylic acid cycle intermediates without clear correlation with the reduced expression of specific E2-OGDH isoforms. Furthermore, the e2-ogdh mutant lines exhibited a reduction by up to 25% in the leaf total amino acids without consistent changes in the amino acid profile. Taken together, our results indicate that the two isoforms of E2 subunit play a similar role in carbon-nitrogen metabolism, in plant growth and in seed weight.
Asunto(s)
Arabidopsis/fisiología , Carbono/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Nitrógeno/metabolismo , Arabidopsis/crecimiento & desarrollo , Regulación hacia Abajo , Regulación de la Expresión Génica de las Plantas , Germinación , Complejo Cetoglutarato Deshidrogenasa/genética , Fotosíntesis , Filogenia , Subunidades de Proteína , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Semillas/enzimología , Semillas/crecimiento & desarrolloRESUMEN
The mitochondrion has emerged as a promising therapeutic target for novel cancer treatments because of its essential role in tumorigenesis and resistance to chemotherapy. Previously, we described a natural compound, 10-((2,5-dihydroxybenzoyl)oxy)decyl) triphenylphosphonium bromide (GA-TPP+C10), with a hydroquinone scaffold that selectively targets the mitochondria of breast cancer (BC) cells by binding to the triphenylphosphonium group as a chemical chaperone; however, the mechanism of action remains unclear. In this work, we showed that GA-TPP+C10 causes time-dependent complex inhibition of the mitochondrial bioenergetics of BC cells, characterized by (1) an initial phase of mitochondrial uptake with an uncoupling effect of oxidative phosphorylation, as previously reported, (2) inhibition of Complex I-dependent respiration, and (3) a late phase of mitochondrial accumulation with inhibition of α-ketoglutarate dehydrogenase complex (αKGDHC) activity. These events led to cell cycle arrest in the G1 phase and cell death at 24 and 48 h of exposure, and the cells were rescued by the addition of the cell-penetrating metabolic intermediates l-aspartic acid ß-methyl ester (mAsp) and dimethyl α-ketoglutarate (dm-KG). In addition, this unexpected blocking of mitochondrial function triggered metabolic remodeling toward glycolysis, AMPK activation, increased expression of proliferator-activated receptor gamma coactivator 1-alpha (pgc1α) and electron transport chain (ETC) component-related genes encoded by mitochondrial DNA and downregulation of the uncoupling proteins ucp3 and ucp4, suggesting an AMPK-dependent prosurvival adaptive response in cancer cells. Consistent with this finding, we showed that inhibition of mitochondrial translation with doxycycline, a broad-spectrum antibiotic that inhibits the 28 S subunit of the mitochondrial ribosome, in the presence of GA-TPP+C10 significantly reduces the mt-CO1 and VDAC protein levels and the FCCP-stimulated maximal electron flux and promotes selective and synergistic cytotoxic effects on BC cells at 24 h of treatment. Based on our results, we propose that this combined strategy based on blockage of the adaptive response induced by mitochondrial bioenergetic inhibition may have therapeutic relevance in BC.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Quinasas de la Proteína-Quinasa Activada por el AMP , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Doxiciclina/farmacología , Sinergismo Farmacológico , Femenino , Gentisatos/química , Gentisatos/farmacología , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Humanos , Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Complejo Cetoglutarato Deshidrogenasa/genética , Mitocondrias/patología , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Proteínas Quinasas/genética , Ribosomas/efectos de los fármacosRESUMEN
KEY MESSAGE: Isoforms of 2-OGDH E1 subunit are not functionally redundant in plant growth and development of A. thaliana. The tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase (2-OGDH) converts 2-oxoglutarate (2-OG) to succinyl-CoA concomitant with the reduction of NAD+. 2-OGDH has an essential role in plant metabolism, being both a limiting step during mitochondrial respiration as well as a key player in carbon-nitrogen interactions. In Arabidopsis thaliana two genes encode for E1 subunit of 2-OGDH but the physiological roles of each isoform remain unknown. Thus, in the present study we isolated Arabidopsis T-DNA insertion knockout mutant lines for each of the genes encoding the E1 subunit of 2-OGDH enzyme. All mutant plants exhibited substantial reduction in both respiration and CO2 assimilation rates. Furthermore, mutant lines exhibited reduced levels of chlorophylls and nitrate, increased levels of sucrose, malate and fumarate and minor changes in total protein and starch levels in leaves. Despite the similar metabolic phenotypes for the two E1 isoforms the reduction in the expression of each gene culminated in different responses in terms of plant growth and seed production indicating distinct roles for each isoform. Collectively, our results demonstrated the importance of the E1 subunit of 2-OGDH in both autotrophic and heterotrophic tissues and suggest that the two E1 isoforms are not functionally redundant in terms of plant growth in A. thaliana.
Asunto(s)
Arabidopsis/enzimología , Carbono/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Nitrógeno/metabolismo , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Complejo Cetoglutarato Deshidrogenasa/genética , Mitocondrias/enzimología , Mutagénesis Insercional , Nitratos/metabolismo , Fenotipo , Filogenia , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Isoformas de Proteínas , Subunidades de Proteína , Plantones/enzimología , Plantones/genética , Plantones/crecimiento & desarrollo , Semillas/enzimología , Semillas/genética , Semillas/crecimiento & desarrolloRESUMEN
Mitochondrial dysfunction has been proposed to play an important role in the neuropathology of glutaric acidemia type I (GA I). However, the relevance of bioenergetics disruption and the exact mechanisms responsible for the cortical leukodystrophy and the striatum degeneration presented by GA I patients are not yet fully understood. Therefore, in the present work we measured the respiratory chain complexes activities I-IV, mitochondrial respiratory parameters state 3, state 4, the respiratory control ratio and dinitrophenol (DNP)-stimulated respiration (uncoupled state), as well as the activities of α-ketoglutarate dehydrogenase (α-KGDH), creatine kinase (CK) and Na+, K+-ATPase in cerebral cortex, striatum and hippocampus from 30-day-old Gcdh-/- and wild type (WT) mice fed with a normal or a high Lys (4.7%) diet. When a baseline (0.9% Lys) diet was given, we verified mild alterations of the activities of some respiratory chain complexes in cerebral cortex and hippocampus, but not in striatum from Gcdh-/- mice as compared to WT animals. Furthermore, the mitochondrial respiratory parameters and the activities of α-KGDH and CK were not modified in all brain structures from Gcdh-/- mice. In contrast, we found a significant reduction of Na(+), K(+)-ATPase activity associated with a lower degree of its expression in cerebral cortex from Gcdh-/- mice. Furthermore, a high Lys (4.7%) diet did not accentuate the biochemical alterations observed in Gcdh-/- mice fed with a normal diet. Since Na(+), K(+)-ATPase activity is required for cell volume regulation and to maintain the membrane potential necessary for a normal neurotransmission, it is presumed that reduction of this enzyme activity may represent a potential underlying mechanism involved in the brain swelling and cortical abnormalities (cortical atrophy with leukodystrophy) observed in patients affected by GA I.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/patología , Encefalopatías Metabólicas/genética , Encefalopatías Metabólicas/patología , Corteza Cerebral/patología , Cuerpo Estriado/patología , Glutaril-CoA Deshidrogenasa/deficiencia , Hipocampo/patología , ATPasa Intercambiadora de Sodio-Potasio/genética , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Animales , Encefalopatías Metabólicas/enzimología , Corteza Cerebral/enzimología , Cuerpo Estriado/enzimología , Creatina Quinasa/genética , Creatina Quinasa/metabolismo , Regulación hacia Abajo , Transporte de Electrón/genética , Alimentos Formulados , Expresión Génica , Glutaril-CoA Deshidrogenasa/genética , Hipocampo/enzimología , Humanos , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismo , Fosforilación Oxidativa , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The tellurium oxyanion tellurite is toxic to most organisms because of its ability to generate oxidative stress. However, the detailed mechanism(s) how this toxicant interferes with cellular processes have yet to be fully understood. As part of our effort to decipher the molecular interactions of tellurite with living systems, we have evaluated the global metabolism of α-ketoglutarate a known antioxidant in Escherichia coli. Tellurite-exposed cells displayed reduced activity of the KG dehydrogenase complex (KGDHc), resulting in increased intracellular KG content. This complex's reduced activity seems to be due to decreased transcription in the stressed cells of sucA, a gene that encodes the E1 component of KGDHc. Furthermore, it was demonstrated that the increase in total reactive oxygen species and superoxide observed upon tellurite exposure was more evident in wild type cells than in E. coli with impaired KGDHc activity. These results indicate that KG may be playing a pivotal role in combating tellurite-mediated oxidative damage.
Asunto(s)
Escherichia coli/efectos de los fármacos , Ácidos Cetoglutáricos/metabolismo , Estrés Oxidativo , Telurio/toxicidad , Dihidrolipoamida Deshidrogenasa/genética , Complejo I de Transporte de Electrón/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Complejo Cetoglutarato Deshidrogenasa/genética , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
It has been assumed that oxidative phosphorylation (OxPhos) in solid tumors is severely reduced due to cytochrome c oxidase substrate restriction, although the measured extracellular oxygen concentration in hypoxic areas seems not limiting for this activity. To identify alternative hypoxia-induced OxPhos depressing mechanisms, an integral analysis of transcription, translation, enzyme activities and pathway fluxes was performed on glycolysis and OxPhos in HeLa and MCF-7 carcinomas. In both neoplasias exposed to hypoxia, an early transcriptional response was observed after 8h (two times increased glycolysis-related mRNA synthesis promoted by increased HIF-1alpha levels). However, major metabolic remodeling was observed only after 24h hypoxia: increased glycolytic protein content (1-5-times), enzyme activities (2-times) and fluxes (4-6-times). Interestingly, in MCF-7 cells, 24h hypoxia decreased OxPhos flux (4-6-fold), and 2-oxoglutarate dehydrogenase and glutaminase activities (3-fold), with no changes in respiratory complexes I and IV activities. In contrast, 24h hypoxia did not significantly affect HeLa OxPhos flux; neither mitochondria related mRNAs, protein contents or enzyme activities, although the enhanced glycolysis became the main ATP supplier. Thus, prolonged hypoxia (a) targeted some mitochondrial enzymes in MCF-7 but not in HeLa cells, and (b) induced a transition from mitochondrial towards a glycolytic-dependent energy metabolism in both MCF-7 and HeLa carcinomas.
Asunto(s)
Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Hipoxia/metabolismo , Mitocondrias/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias de la Mama/patología , Carcinoma/patología , Complejo I de Transporte de Electrón/metabolismo , Metabolismo Energético , Femenino , Glutaminasa/genética , Glutaminasa/metabolismo , Glucólisis , Células HeLa , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Fosforilación Oxidativa , Neoplasias del Cuello Uterino/patologíaRESUMEN
Phenoxyalkanoic herbicides such as 2,4-dichlorophenoxyacetate (2,4-D), 2,4-dichlorophenoxybutyrate (2,4-DB) or mecoprop are widely used to control broad-leaf weeds. Several bacteria have been reported to degrade these herbicides using the α-ketoglutarate-dependent, 2,4-dichlorophenoxyacetate dioxygenase encoded by the tfdA gene, as the enzyme catalysing the first step in the catabolic pathway. The effects of exposure to different phenoxyalkanoic herbicides in the soil bacterial community and in the tfdA genes diversity were assessed using an agricultural soil exposed to these anthropogenic compounds. Total community bacterial DNA was analysed by terminal restriction fragment length polymorphism of the 16S rRNA and the tfdA gene markers, and detection and cloning of tfdA gene related sequences, using PCR primer pairs. After up to 4 months of herbicide exposure, significant changes in the bacterial community structure were detected in soil microcosms treated with mecoprop, 2,4-DB and a mixture of both plus 2,4-D. An impressive variety of novel tfdA gene related sequences were found in these soil microcosms, which cluster in new tfdA gene related sequence groups, unequally abundant depending on the specific herbicide used in soil treatment. Structural analysis of the putative protein products showed small but significant amino acid differences. These tfdA gene sequence variants are, probably, required for degradation of natural substrate(s) structurally related to these herbicides and their presence explains self-remediation of soils exposed to phenoxyalkanoic herbicides.