RESUMEN
Rhein, a monomeric anthraquinone obtained from the plant herb species Polygonum multiflorum and P. cuspidatum, has been proposed to have anticancer activity. This activity has been suggested to be associated with mitochondrial injury due to the induction of mitochondrial permeability transition pore (mPTP) opening. In this study, the effects of 5-80 µM rhein on cell viability, half-maximal inhibitory concentration (IC50 value), resistance index, and apoptosis were assessed in the liver cancer cell lines SMMC-7721 and SMMC-7721/DOX (doxorubicin-resistant cells). Rhein (10-80 µM) significantly reduced the viability of both cell lines; 20 µM rhein significantly increased sensitivity to DOX and increased apoptosis in SMMC-7721 cells, but reversed resistance to DOX by 7.24-fold in SMMC-7721/DOX cells. Treatment with rhein increased accumulation of DOX in SMMC-7721/DOX cells, inhibited mitochondrial energy metabolism, decreased cellular ATP, and ADP levels, and altered the ratio of ATP to ADP. These effects may result from the binding of rhein with voltage-dependent ion channels (VDACs), adenine nucleotide translocase (ANT), and cyclophilin D, affecting their function and leading to the inhibition of ATP transport by VDACs and ANT. ATP synthesis was greatly reduced and mitochondrial inner membrane potential decreased. Together, these results indicate that rhein could reverse drug resistance in SMMC-7721/DOX cells by inhibiting energy metabolism and inducing mPTP opening. © 2018 BioFactors, 45(1):85-96, 2019.
Asunto(s)
Antraquinonas/farmacología , Antibióticos Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Adenosina Trifosfato/antagonistas & inhibidores , Adenosina Trifosfato/biosíntesis , Antraquinonas/aislamiento & purificación , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclofilinas/genética , Ciclofilinas/metabolismo , Combinación de Medicamentos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Fallopia japonica/química , Fallopia multiflora/química , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/genética , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/genética , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Extractos Vegetales/química , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
The importance of a plastidial soluble inorganic pyrophosphatase (psPPase) and an ATP/ADP translocator (NTT) for starch composition and tuber formation in potato (Solanum tuberosum) was evaluated by individual and simultaneous down-regulation of the corresponding endogenous genes. Starch and amylose content of the transgenic lines were considerably lower, and granule size substantially smaller, with down-regulation of StpsPPase generating the most pronounced effects. Single-gene down-regulation of either StpsPPase or StNTT resulted in increased tuber numbers per plant and higher fresh weight yield. In contrast, when both genes were inhibited simultaneously, some lines developed only a few, small and distorted tubers. Analysis of metabolites revealed altered amounts of sugar intermediates, and a substantial increase in ADP-glucose content of the StpsPPase lines. Increased amounts of intermediates of vitamin C biosynthesis were also observed. This study suggests that hydrolysis of pyrophosphate (PPi) by action of a psPPase is vital for functional starch accumulation in potato tubers and that no additional mechanism for consuming, hydrolysing, or exporting PPi exists in the studied tissue. Additionally, it demonstrates that functional PPi hydrolysis in combination with efficient ATP import is essential for tuber formation and development.
Asunto(s)
Pirofosfatasa Inorgánica/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/crecimiento & desarrollo , Plastidios/enzimología , Solanum tuberosum/enzimología , Almidón/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Regulación de la Expresión Génica de las Plantas , Pirofosfatasa Inorgánica/genética , Translocasas Mitocondriales de ADP y ATP/genética , Proteínas de Plantas/genética , Tubérculos de la Planta/enzimología , Tubérculos de la Planta/genética , Tubérculos de la Planta/metabolismo , Plastidios/genética , Solanum tuberosum/genética , Solanum tuberosum/crecimiento & desarrollo , Solanum tuberosum/metabolismoRESUMEN
Capsules, powders and tablets containing raw coffee extract are advertised to the consumer as antioxidant rich dietary supplements as part of a healthy diet. We isolated carboxyatractyligenin (4), 2-O-ß-d-glucopyranosyl carboxyatractyligenin (6) and 3'-O-ß-d-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-carboxyatractyligenin)-ß-d-glucopyranoside (8) from green coffee and found strong inhibitory effects on phosphorylating respiration in isolated mitochondria similar to the effects of the known phytotoxin carboxyatractyloside. LC-MS/MS analysis of commercial green coffee based dietary supplements revealed the occurrence of carboxyatractyligenin, 3'-O-ß-d-glucopyranosyl-2'-O-isovaleryl-2ß-(2-desoxy-carboxyatractyligenin)-ß-d-glucopyranoside, and 2-O-ß-d-glucopyranosyl carboxyatractyligenin in concentrations up to 4.0, 5.7, and 41.6µmol/g, respectively. These data might help to gain first insight into potential physiological side-effects of green coffee containing dietary supplement.
Asunto(s)
Atractilósido/análogos & derivados , Café/química , Suplementos Dietéticos , Translocasas Mitocondriales de ADP y ATP/metabolismo , Animales , Antioxidantes/farmacología , Atractilósido/farmacología , Cromatografía Liquida , Manipulación de Alimentos , Masculino , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Translocasas Mitocondriales de ADP y ATP/antagonistas & inhibidores , Fosforilación , Polvos/química , Espectrometría de Masas en TándemRESUMEN
The combination of isotretinoin (13-cis-retinoic acid) with antiestrogens seems to be a promising strategy for cancer chemotherapy. The aim of the study was to evaluate the effects of isotretinoin alone or in combination with 4-hydroxytamoxifen (OHTAM) and with its prodrug tamoxifen (TAM), on the functions of rat liver mitochondria, i.e., mitochondrial permeability transition (MPT), bioenergetic functions and adenine nucleotide translocase (ANT). Isotretinoin (5 nmol/mg protein) induced the Ca²âº-dependent MPT pore opening in mitochondria energized with succinate, which was prevented by OHTAM, cyclosporine A, TAM and ANT ligands. When mitochondria were energized with glutamate/malate and in the absence of added Ca²âº isotretinoin decreased the state 3 respiration, the ATP levels, the active ANT content and increased the lag phase of the phosphorylation cycle, demonstrating that isotretinoin decreased the mitochondrial phosphorylation efficiency. These changes of isotretinoin in bioenergetic parameters were not significant in the presence of succinate. The effects of isotretinoin at 5 nmol/mg protein on the Ca²âº-dependent MPT and phosphorylative efficacy may be related with interactions with the ANT. Above 10 nmol/mg protein isotretinoin strongly diminished the active ANT content, decreased the Δψ, inhibited the complex I and induced proton leak through the Fo fraction of complex V. The combination of OHTAM with isotretinoin only induced significant changes in the energy production systems at concentrations ≥5 nmol isotretinoin/mg protein. Therefore, our results suggest that isotretinoin-associated liver toxicity is possibly related with mitochondrial dysfunctions and that the combination with OHTAM may contribute to decrease its toxicity.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Moduladores de los Receptores de Estrógeno/farmacología , Isotretinoína/farmacología , Mitocondrias Hepáticas/efectos de los fármacos , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacología , Animales , Antineoplásicos Hormonales/administración & dosificación , Permeabilidad de la Membrana Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Metabolismo Energético , Moduladores de los Receptores de Estrógeno/administración & dosificación , Isotretinoína/administración & dosificación , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Fosforilación Oxidativa , Ratas , Ratas Wistar , Tamoxifeno/administración & dosificaciónRESUMEN
Acitretin is a synthetic retinoid used for severe extensive psoriasis and it has been shown to be an effective and a safe therapeutic drug for other diseases including cancer when used in combination with other agents. However, cases of acitretin-associated liver injury have been documented, but the possible mechanisms of acitretin-associated hepatotoxicity and apoptosis are not entirely clarified. This study reports that mitochondrial dysfunctions may play an important role in liver injury and apoptosis induced by this retinoid. Acitretin (5-20 µM) impaired mitochondrial phosphorylation efficiency as demonstrated by the decrease in the state 3 respiration and ATP levels, and by the increase in the lag phase of ADP phosphorylation cycle, without affecting the membrane potential. Acitretin induced Ca(2+)-mediated mitochondrial permeability transition (MPT) and decreased the adenine nucleotide translocase (ANT) content. Acitretin-induced MPT was not prevented by thiol group protecting and antioxidant agents, excluding the involvement of oxidative stress mechanisms. However, MPT was prevented by ANT ligands ATP, ADP, tamoxifen and 4-hydroxytamoxifen, implying that the MPT induction by acitretin is mediated by the ANT. ANT plays a major role in promoting apoptosis and ATP synthesis, and it is still considered as a structural component of the pore with a regulatory role in MPT formation. Therefore, our results, including the decrease in the state 3 respiration and the increase in the lag phase of phosphorylation cycle, the ATP depletion and the induction of Ca(2+)-mediated MPT, indicate that acitretin-associated liver toxicity and apoptosis is possibly related with mitochondrial dysfunctions due to interactions with the ANT. Additionally, the combination of acitretin with other drugs, such as antiestrogens, which are able to inhibit the MPT, may contribute to decrease the toxicity induced by acitretin.
Asunto(s)
Acitretina/toxicidad , Apoptosis/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Animales , Metabolismo Energético/efectos de los fármacos , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Wistar , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologíaRESUMEN
Mitochondria from diabetic hearts are sensitized to mitochondrial permeability transition pore (PTP) opening, which may be responsible for the increased propensity for cardiac injury in diabetic hearts. The purpose of this study was to determine if redox-dependent PTP opening contributes to augmented injury in diabetic hearts, and if compounds targeted at mitochondrial PTP, ROS, and calcium influx protected diabetic hearts from injury. Hearts from control or streptozotocin-induced diabetic rats were excised for either whole-heart or isolated mitochondria experiments. Myocardial glutathione content was oxidized in diabetic hearts when compared to control, and this translated to increased oxidation of the adenine nucleotide translocase in diabetic hearts. Diabetic mitochondria displayed significantly greater sensitivity to PTP opening than non-diabetic counterparts, which was reversed with the thiol-reducing agent dithiothreitol. The thiol-oxidant diamide increased calcium sensitivity in control, but not diabetic mitochondria. Diabetic animals treated with the mitochondria-targeted ROS suppressing peptide MTP-131 also showed improved resistance to PTP opening. In separate experiments hearts underwent ex vivo ischemia/reperfusion (IR). Diabetic hearts were more susceptible to IR injury, with infarct sizes of 60 ± 4% of the area-at-risk (vs. 46 ± 2% in non-diabetics; P<0.05). Administration of the PTP blocker NIM811 (5 µM), MTP-131 (1 nM) or the mitochondrial calcium uniporter blocker minocycline (1 µM) at the onset of reperfusion reduced infarct sizes in both control and diabetic hearts. These findings suggest that augmented susceptibility to injury in the diabetic heart is mediated by redox-dependent shifts in PTP opening, and that three novel mitochondria-targeted agents administered at reperfusion may be suitable adjuvant reperfusion therapies to attenuate injury in diabetic patients.
Asunto(s)
Calcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Oligopéptidos/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/uso terapéutico , Señalización del Calcio , Cardiotónicos/farmacología , Cardiotónicos/uso terapéutico , Ciclosporina/farmacología , Ciclosporina/uso terapéutico , Diabetes Mellitus Experimental/complicaciones , Glutatión/metabolismo , Disulfuro de Glutatión/metabolismo , Corazón/efectos de los fármacos , Corazón/fisiopatología , Hemodinámica , Técnicas In Vitro , Masculino , Minociclina/farmacología , Minociclina/uso terapéutico , Mitocondrias Cardíacas/efectos de los fármacos , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/antagonistas & inhibidores , Poro de Transición de la Permeabilidad Mitocondrial , Daño por Reperfusión Miocárdica/etiología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/prevención & control , Oligopéptidos/uso terapéutico , Oxidación-Reducción , Permeabilidad , Ratas , Ratas Sprague-DawleyRESUMEN
There is an increased need for high-yield protein production platforms to meet growing demand. Tuber-based production in Solanum tuberosum offers several advantages, including high biomass yield, although protein concentration is typically low. In this work, we investigated the question whether minor interruption of starch biosynthesis can have a positive effect on tuber protein content and/or tuber biomass, as previous work suggested that partial obstruction of starch synthesis had variable effects on tuber yield. To this end, we used a RNAi approach to knock down ATP/ADP transporter and obtained a large number of transgenic lines for screening of lines with improved tuber protein content and/or tuber biomass. The initial screening was based on tuber biomass because of its relative simplicity. We identified a line, riAATP1-10, with minor (less than 15%) reduction in starch, that had a nearly 30% increase in biomass compared to wild-type, producing both more and larger tubers with altered morphological features compared to wild-type. riAATP1-10 tubers have a higher concentration of soluble protein compared to wild-type tubers, with nearly 50% more soluble protein. We assessed the suitability of this line as a new bioreactor by expressing a human scFv, reaching over 0.5% of total soluble protein, a 2-fold increase over the highest accumulating line in a wild-type background. Together with increased biomass and increased levels in total protein content, foreign protein expression in riAATP1-10 line would translate into a nearly 4-fold increase in recombinant protein yield per plant. Our results indicate that riAATP1-10 line provides an improved expression system for production of foreign proteins.
Asunto(s)
Translocasas Mitocondriales de ADP y ATP/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Interferencia de ARN , Solanum tuberosum/genética , Secuencia de Bases , Biomasa , Reactores Biológicos , Biotecnología , Electroforesis en Gel de Poliacrilamida , Técnicas de Silenciamiento del Gen , Humanos , Translocasas Mitocondriales de ADP y ATP/deficiencia , Translocasas Mitocondriales de ADP y ATP/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/química , Tubérculos de la Planta/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Solanum tuberosum/metabolismo , Almidón/metabolismoRESUMEN
The SCaMCs (small calcium-binding mitochondrial carriers) constitute a subfamily of mitochondrial carriers responsible for the ATP-Mg/P(i) exchange with at least three paralogues in vertebrates. SCaMC members are proteins with two functional domains, the C-terminal transporter domain and the N-terminal domain which harbours calcium-binding EF-hands and faces the intermembrane space. In the present study, we have characterized a shortened fourth paralogue, SCaMC-3L (SCaMC-3-like; also named slc25a41), which lacks the calcium-binding N-terminal extension. SCaMC-3L orthologues are found exclusively in mammals, showing approx. 60% identity to the C-terminal half of SCaMC-3, its closest paralogue. In mammalian genomes, SCaMC-3 and SCaMC-3L genes are adjacent on the same chromosome, forming a head-to-tail tandem array, and show identical exon-intron boundaries, indicating that SCaMC-3L could have arisen from an SCaMC-3 ancestor by a partial duplication event which occurred prior to mammalian radiation. Expression and functional data suggest that, following the duplication event, SCaMC-3L has acquired more restrictive functions. Unlike the broadly expressed longer SCaMCs, mouse SCaMC-3L shows a limited expression pattern; it is preferentially expressed in testis and, at lower levels, in brain. SCaMC-3L transport activity was studied in yeast deficient in Sal1p, the calcium-dependent mitochondrial ATP-Mg/P(i) carrier, co-expressing SCaMC-3L and mitochondrial-targeted luciferase, and it was found to perform ATP-Mg/P(i) exchange, in a similar manner to Sal1p or other ATP-Mg/P(i) carriers. However, metabolite transport through SCaMC-3L is calcium-independent, representing a novel mechanism involved in adenine nucleotide transport across the inner mitochondrial membrane, different to ADP/ATP translocases or long SCaMC paralogues.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas de Transporte de Anión/metabolismo , Magnesio/metabolismo , Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Fósforo/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/genética , Encéfalo/metabolismo , Calcio/metabolismo , Línea Celular , Chlorocebus aethiops , Regulación de la Expresión Génica , Humanos , Masculino , Ratones , Translocasas Mitocondriales de ADP y ATP/genética , Modelos Moleculares , Datos de Secuencia Molecular , Especificidad de Órganos , Filogenia , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Testículo/metabolismoRESUMEN
Plasmodium falciparum mitochondrial ADP/ATP transporter or adenylate translocase (PfAdT) was previously characterised at the molecular level and intracellularly located by immuno-electromicroscopy. Inhibition of this transporter blocks parasite development in erythrocytes. In this study, PfAdT was expressed in C43 (DE3) Escherichia coli strain under isopropyl beta-d-thiogalacto-pyranoside (IPTG) induction to screen inhibitory molecules. PfAdT was integrated directly into the bacterial cytoplasmic membrane. Whereas IPTG-induced bacterial cells imported radioactively labelled ATP, non-induced cells did not. The transporter bound specifically ADP and ATP, but not AMP. IPTG-induced cells preloaded with labelled ATP exported ATP after exogenous addition of unlabelled ADP or ATP, indicating a counter exchange transport mechanism. Bongrekic acid and atractyloside, two well-known specific inhibitors of mitochondrial ADP/ATP transporter, were tested. This experimental model was evaluated using three Malagasy crude plants extracts which have shown antiplasmodial activity on in vitro parasite cultures.
Asunto(s)
Antimaláricos/farmacología , Translocasas Mitocondriales de ADP y ATP/antagonistas & inhibidores , Translocasas Mitocondriales de ADP y ATP/metabolismo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Animales , Atractilósido/farmacología , Western Blotting , Ácido Bongcréquico/farmacología , Membrana Celular/enzimología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Eritrocitos/parasitología , Escherichia coli/enzimología , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Translocasas Mitocondriales de ADP y ATP/genética , Extractos Vegetales/farmacología , Plasmodium falciparum/crecimiento & desarrollo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Three Annonaceous acetogenins exhibited in vitro antimalarial activities on a chloroquine-resistant Plasmodium falciparum strain, with IC50s ranging from 5 to 10 microM. Structure-activity relationships showed that maximal antimalarial activity occurred in the presence of at least one tetrahydrofuran moiety and a synergistic action with chloroquine was observed. These acetogenins partially inhibited the P. falciparum adenylate translocase.
Asunto(s)
Aminoquinolinas/farmacología , Antimaláricos/farmacología , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Alcoholes Grasos/farmacología , Lactonas/farmacología , Extractos Vegetales/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Acetogeninas , Aminoquinolinas/química , Animales , Antimaláricos/química , Farmacorresistencia Microbiana , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/microbiología , Pruebas de Sensibilidad Microbiana , Translocasas Mitocondriales de ADP y ATP/antagonistas & inhibidores , Translocasas Mitocondriales de ADP y ATP/metabolismo , Plasmodium falciparum/aislamiento & purificación , Sensibilidad y Especificidad , Relación Estructura-ActividadRESUMEN
Tubers of transgenic potato (Solanum tuberosum) plants with decreased activity of the plastidic ATP/ADP transporter AATP1 display reduced levels of starch, modified tuber morphology, and altered concentrations of primary metabolites. Here, we demonstrate that the spontaneous production of hydrogen peroxide, the endogenous content of salicylic acid, and the levels of mRNAs of various defense-related genes are similar in tuber discs of wild-type and AATP1(St) antisense plants. However, upon challenging the tissue with fungal elicitors or culture supernatants of the soft rot-causing pathogen Erwinia carotovora subsp. atroseptica, the AATP1(St) antisense tubers exhibit highly potentiated activation of defense responses when compared with wild-type tissue. The augmented defense responses comprise enhanced accumulation of transcripts of five defense-related genes (beta-1,3-GLUCANASE B2 and A1, CHITINASE B3 and A2, and Phe AMMONIA-LYASE) and enhanced elicitation (up to 21-fold) of the early hydrogen peroxide burst. The potentiated activation of cellular defense responses in AATP1(St) antisense tubers is not accompanied by a precedent increase in endogenous salicylic acid levels, but is associated with a strongly enhanced resistance of the tissue to E. carotovora. From these results, we conclude that inhibition of primary metabolic reactions induces a primed state that sensitizes the potato tubers for improved elicitation of various cellular defense responses, which likely contribute to enhanced E. carotovora resistance.
Asunto(s)
Translocasas Mitocondriales de ADP y ATP/metabolismo , Pectobacterium carotovorum/crecimiento & desarrollo , Tallos de la Planta/fisiología , Plastidios/metabolismo , Solanum tuberosum/fisiología , ADN sin Sentido/genética , Peróxido de Hidrógeno/metabolismo , Inmunidad Innata , Translocasas Mitocondriales de ADP y ATP/genética , Enfermedades de las Plantas/microbiología , Tallos de la Planta/microbiología , ARN Mensajero/metabolismo , Ácido Salicílico/metabolismo , Solanum tuberosum/genética , Solanum tuberosum/microbiologíaRESUMEN
In order to gain some insight into mitochondria permeability under water stress, intact coupled mitochondria were isolated from water stress adapted potato cells and investigations were made of certain transport processes including the succinate/malate and ADP/ATP exchanges, the plant mitochondrial ATP-sensitive potassium channel (PmitoKATP) and the plant uncoupling mitochondrial protein (PUMP). The Vmax values measured for succinate/malate and ADP/ATP carriers, as photometrically investigated, as well as the same values for the PmitoK(ATP) and the PUMP were found to increase; this suggested that mitochondria adaptation to water stress can cause an increase in the membrane permeability.
Asunto(s)
Metabolismo Energético/fisiología , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Solanum tuberosum/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Metabolismo Energético/efectos de los fármacos , Membranas Intracelulares/efectos de los fármacos , Canales Iónicos , Malatos/metabolismo , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Translocasas Mitocondriales de ADP y ATP/efectos de los fármacos , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas Mitocondriales , Modelos Biológicos , Permeabilidad , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Solanum tuberosum/citología , Solanum tuberosum/efectos de los fármacos , Ácido Succínico/metabolismo , Proteína Desacopladora 1 , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Deletions of the mitochondrial DNA (mtDNA) have been shown to accumulate with age in a variety of species regardless of mean or maximal life span. This implies that such mutations are either a molecular biomarker of senescence or that they are more causally linked to senescence itself. One assay that can be used to detect these mtDNA mutations is the long-extension polymerase chain reaction assay. This assay amplifies approximately 16 kb of the mtDNA in mammalian mitochondria and preferentially amplifies mtDNAs that are either deleted or duplicated. We have applied this assay to the aging human brain and found a heterogeneous array of rearranged mtDNAs. In addition, we have developed in situ polymerase chain reaction to detect mtDNA within individual cells of both the mouse and the human brain as a first step in identifying and enumerating cells containing mutant mtDNAs in situ.
Asunto(s)
Envejecimiento/genética , ADN Mitocondrial/genética , Reordenamiento Génico , Mitocondrias/enzimología , Anciano , Animales , Cerebelo/metabolismo , Cartilla de ADN , Metabolismo Energético/fisiología , Femenino , Lóbulo Frontal/metabolismo , Eliminación de Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/genética , Translocasas Mitocondriales de ADP y ATP/genética , Translocasas Mitocondriales de ADP y ATP/metabolismo , Estrés Oxidativo/fisiología , Reacción en Cadena de la Polimerasa/métodos , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismoRESUMEN
The mitochondrial ADP/ATP translocator, also called adenine nucleotide translocase (ANT), is synthesized in plants with an N-terminal extension which is cleaved upon import into mitochondria. In contrast, the homologous proteins of mammals or fungi do not contain such a transient amino terminal presequence. To investigate whether the N-terminal extension is needed for correct intracellular sorting in vivo, translational fusions were constructed of the translocator cDNA--with and without presequence--with the beta-glucuronidase (gus) reporter gene. The distribution of reporter enzymatic activity in the subcellular compartments of transgenic plants and transformed yeast cells was subsequently analysed. The results show that: (i) the plant translocator presequence is not necessary for the correct localization of the ANT to the mitochondria; (ii) the mitochondrial targeting information contained in the mature part of the protein is sufficient to overcome, to some extent, the presence of plastid transit peptides; and (iii) the presequence alone is not able to target a passenger protein to mitochondria in vivo.
Asunto(s)
Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Señales de Clasificación de Proteína/metabolismo , Solanum tuberosum/metabolismo , Secuencia de Bases , ADN de Plantas , Glucuronidasa/genética , Translocasas Mitocondriales de ADP y ATP/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Solanum tuberosum/genéticaRESUMEN
A 42-kDa plant outer mitochondrial membrane protein, MOM42, has been identified as an essential component of the plant mitochondrial precursor protein translocation apparatus. Immunological cross-reactivity has been detected between antibodies raised against both Neurospora and yeast mitochondrial outer membrane proteins and plant mitochondrial outer membrane proteins. Immunocompetition studies showed that import of precursors to Rieske FeS protein, ATPase su9-DHFR, and the adenine nucleotide transporter was inhibited in the presence of antibody to MOM42. The inhibition of Rieske Fes and su9-DHFR import was greater than that of the adenine nucleotide transporter. The competition studies suggest that the MOM42 is involved in the translocation of bound precursor proteins. The import data and the Western blots suggest that components of the mitochondrial import system are highly conserved.
Asunto(s)
Proteínas Portadoras/metabolismo , Complejo III de Transporte de Electrones , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Mitocondrias/metabolismo , Proteínas de Plantas , Precursores de Proteínas/metabolismo , Verduras/metabolismo , Adenosina Trifosfatasas/metabolismo , Transporte Biológico , Western Blotting , Proteínas Portadoras/inmunología , Reacciones Cruzadas , Fabaceae/metabolismo , Membranas Intracelulares/química , Proteínas Hierro-Azufre/metabolismo , Proteínas de la Membrana/inmunología , Translocasas Mitocondriales de ADP y ATP/metabolismo , Plantas Medicinales , Especificidad de la EspecieRESUMEN
Carboxyatractyloside partially restored the transmembrane electrical potential difference (delta psi) dissipated by low concentrations of palmitate in pea stem mitochondria. This effect was more marked when mitochondria from sunflower were assayed. It is suggested that the ATP/ADP translocator is involved in the free fatty acid-induced uncoupling of oxidative phosphorylation in plant mitochondria, only when its level is sufficiently high and the concentration of the fatty acid is low to collapse only partially the delta psi.
Asunto(s)
Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Ácidos Palmíticos/farmacología , Plantas/ultraestructura , Desacopladores/farmacología , Atractilósido/análogos & derivados , Atractilósido/farmacología , Fabaceae , Helianthus , Potenciales de la Membrana/efectos de los fármacos , NAD/farmacología , Consumo de Oxígeno/efectos de los fármacos , Ácido Palmítico , Plantas/metabolismo , Plantas MedicinalesRESUMEN
Carboxyatractyloside partially restored the transmembrane electrical potential difference (delta psi) dissipated by low concentrations of palmitate in pea stem mitochondria. This effect was more marked when mitochondria from sunflower were assayed. It is suggested that the ATP/ADP translocator is involved in the free fatty acid-induced uncoupling of oxidative phosphorylation in plant mitochondria, only when its level is sufficiently high and the concentration of the fatty acid is low to collapse only partially delta psi.
Asunto(s)
Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Ácidos Palmíticos/farmacología , Plantas/ultraestructura , Desacopladores/farmacología , Atractilósido/análogos & derivados , Atractilósido/farmacología , Fabaceae , Helianthus , Potenciales de la Membrana/efectos de los fármacos , NAD/farmacología , Consumo de Oxígeno/efectos de los fármacos , Ácido Palmítico , Plantas/metabolismo , Plantas MedicinalesRESUMEN
Two maize genes and cDNAs encoding the mitochondrial adenine nucleotide translocator (ANT), a nuclear-encoded inner mitochondrial membrane carrier protein, have previously been isolated in this laboratory. Sequence analysis revealed the existence of much longer open reading frames than the corresponding fungal and mammalian ANT genes. Potato ANT cDNAs have subsequently been isolated and sequenced and alignment of the deduced plant amino acid sequences with the equivalent fungal and mammalian polypeptides indicated that the plant proteins contain N-terminal extensions. When the plant cDNA clones are expressed in vitro they direct the synthesis of precursor proteins that are specifically processed at the N-terminus upon import into isolated mitochondria. N-terminal amino acid sequence data obtained from the native proteins purified from both maize and potato mitochondria has allowed identification of the putative processing sites. Further import analysis has shown that two distinct regions of the maize precursor protein contain targeting information, the 97 amino acids at the N-terminus and the 267 C-terminal amino acids. This is the first report that provides experimental evidence that the adenine nucleotide translocator of higher plants is synthesized as a large precursor protein that is specifically cleaved upon import into mitochondria. Import of ANT into higher plant mitochondria therefore appears to be different to the corresponding process in fungal and mammalian systems where targeting of ANT to mitochondria is mediated by internal signals and there is no N-terminal processing.
Asunto(s)
Mitocondrias/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína/metabolismo , Zea mays/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Sistema Libre de Células , Análisis Mutacional de ADN , Eliminación de Gen , Translocasas Mitocondriales de ADP y ATP/genética , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Relación Estructura-Actividad , Fracciones Subcelulares , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Zea mays/genéticaRESUMEN
Linolenic, linoleic, oleic, palmitic and stearic acids (FFA) collapse the electrical potential of pea stem mitochondria in the absence or in the presence of 0.5 mM Mg2+. Higher concentrations of this cation (5 mM) lower the rate of dissipation caused by linoleic, oleic and palmitic acids, while abolishing that induced by stearic acid. Carboxyatractyloside and ADP do not reverse the FFA-induced collapse both in the presence or absence of Mg2+. EDTA, EGTA or BHT do not influence the dissipation caused by FFA that, in addition, is not linked to lipid peroxidation evaluated as malondialdehyde or conjugated diene formation. Only linolenic acid sustains a peroxidation which, however, appears to be caused by its own oxidation catalysed by lipoxygenases rather than by membrane lipoperoxidation induced by this free fatty acid. These results suggest that neither the ATP/ADP exchanger nor lipid peroxidation appear to be involved in FFA-induced uncoupling in pea stem mitochondria.
Asunto(s)
Fabaceae/fisiología , Ácidos Grasos no Esterificados/fisiología , Mitocondrias/metabolismo , Plantas Medicinales , Cationes Bivalentes , Membranas Intracelulares/metabolismo , Peroxidación de Lípido , Magnesio/metabolismo , Potenciales de la Membrana , Translocasas Mitocondriales de ADP y ATP/metabolismo , NAD/metabolismoRESUMEN
The ADP/ATP translocator is an abundant protein of the mitochondrial inner membrane, which in fungi and mammals is synthesized without a presequence. Here we report that the translocator from potato has an amino-terminal extension which may function in mitochondrial targeting. Several cDNA clones encoding the nucleotide sequence of the ADP/ATP translocator have been isolated from potato leaf and tuber cDNA libraries constructed in lambda phages. Only one class of cDNA clones was found but possibly different translocator genes are expressed in other tissues. High levels of transcripts for the translocator are found in all tissues analysed. Sequence determination of the complete insert of one of the clones reveals a long open reading frame of 1158 bp encoding a protein of 386 amino acids corresponding to a calculated molecular weight of 42 kDa. In contrast, the ADP/ATP translocator proteins from fungi and mammals are significantly smaller. Comparison of the Neurospora translocator with the potato protein shows about 75% sequence homology, being confined to the region after amino acid 85 of the potato polypeptide. Antibodies directed against the fungal translocator recognize a protein of 30 kDa in the inner membrane of potato mitochondria, suggesting that the mature protein has a similar size as the translocators from fungi and mammals. Thus, the additional segment of the potato ADP/ATP translocator forms an amino-terminal extension which may be involved in the import of the protein into plant mitochondria.