RESUMO
Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria from plant cells for physiological and biochemical analyses is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis (Arabidopsis thaliana) with a triple hemagglutinin tag for rapid purification via a single affinity-purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant deficient in the mitochondrial Glu transporter À BOUT DE SOUFFLE (BOU) and identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 controls. Our work sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.
Assuntos
Arabidopsis/metabolismo , Cromatografia de Afinidade/métodos , Mitocôndrias , Plântula/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glutamina/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Proteínas de Membrana Transportadoras/genética , Microscopia Confocal , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fragmentos de Peptídeos/genética , Plantas Geneticamente Modificadas , Proteoma/genética , Proteoma/metabolismo , Proteômica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cardiotoxicity is a highly relevant, because often life-threatening, adverse effect of doxorubicin (Doxo)-based anticancer therapy. Here, we investigated the Doxo-response of cardiovascular stem/progenitor cells employing a mouse embryonic stem cell (mESC)-based in vitro differentiation model. Endothelial progenitor cells revealed a pronounced Doxo sensitivity as compared to mESC, differentiated endothelial-like (EC) and cardiomyocyte-like cells (CM) and CM progenitors, which rests on the activation of senescence. Doxo treatment of EC progenitors altered protein expression of individual endothelial markers, actin cytoskeleton morphology, mRNA expression of genes related to mitochondrial functions, autophagy, apoptosis, and DNA repair as well as mitochondrial DNA content, respiration and ATP production in the surviving differentiated EC progeny. By contrast, LDL uptake, ATP-stimulated Ca2+ release, and cytokine-stimulated ICAM-1 expression remained unaffected by the anthracycline treatment. Thus, exposure of EC progenitors to Doxo elicits isolated and persistent dysfunctions in the surviving EC progeny. In conclusion, we suggest that Doxo-induced injury of EC progenitors adds to anthracycline-induced cardiotoxicity, making this cell-type a preferential target for pharmacoprotective and regenerative strategies.
Assuntos
Cardiotoxicidade/genética , Doxorrubicina/efeitos adversos , Células Progenitoras Endoteliais/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/genética , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cardiotoxicidade/patologia , Cardiotoxicidade/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Neoplasias/complicações , Neoplasias/tratamento farmacológicoRESUMO
Astrocyte dysfunction is a primary factor in hepatic encephalopathy (HE) impairing neuronal activity under hyperammonemia. In particular, the early events causing ammonia-induced toxicity to astrocytes are not well understood. Using established cellular HE models, we show that mitochondria rapidly undergo fragmentation in a reversible manner upon hyperammonemia. Further, in our analyses, within a timescale of minutes, mitochondrial respiration and glycolysis were hampered, which occurred in a pH-independent manner. Using metabolomics, an accumulation of glucose and numerous amino acids, including branched chain amino acids, was observed. Metabolomic tracking of 15N-labeled ammonia showed rapid incorporation of 15N into glutamate and glutamate-derived amino acids. Downregulating human GLUD2 [encoding mitochondrial glutamate dehydrogenase 2 (GDH2)], inhibiting GDH2 activity by SIRT4 overexpression, and supplementing cells with glutamate or glutamine alleviated ammonia-induced inhibition of mitochondrial respiration. Metabolomic tracking of 13C-glutamine showed that hyperammonemia can inhibit anaplerosis of tricarboxylic acid (TCA) cycle intermediates. Contrary to its classical anaplerotic role, we show that, under hyperammonemia, GDH2 catalyzes the removal of ammonia by reductive amination of α-ketoglutarate, which efficiently and rapidly inhibits the TCA cycle. Overall, we propose a critical GDH2-dependent mechanism in HE models that helps to remove ammonia, but also impairs energy metabolism in mitochondria rapidly.