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1.
Am J Physiol Cell Physiol ; 309(2): C81-91, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25994788

RESUMO

Excessive oxidation is widely accepted as a precursor to deleterious cellular function. On the other hand, an awareness of the role of reductive stress as a similar pathological insult is emerging. Here we report early dynamic changes in compartmentalized glutathione (GSH) redox potentials in living cells in response to exogenously supplied thiol-based antioxidants. Noninvasive monitoring of intracellular thiol-disulfide exchange via a genetically encoded biosensor targeted to cytosol and mitochondria revealed unexpectedly rapid oxidation of the mitochondrial matrix in response to GSH ethyl ester or N-acetyl-l-cysteine. Oxidation of the probe occurred within seconds in a concentration-dependent manner and was attenuated with the membrane-permeable ROS scavenger tiron. In contrast, the cytosolic sensor did not respond to similar treatments. Surprisingly, the immediate mitochondrial oxidation was not abrogated by depolarization of mitochondrial membrane potential or inhibition of mitochondrial GSH uptake. After detection of elevated levels of mitochondrial ROS, we systematically inhibited multisubunit protein complexes of the mitochondrial respiratory chain and determined that respiratory complex III is a downstream target of thiol-based compounds. Disabling complex III with myxothiazol completely blocked matrix oxidation induced with GSH ethyl ester or N-acetyl-l-cysteine. Our findings provide new evidence of a functional link between exogenous thiol-containing antioxidants and mitochondrial respiration.


Assuntos
Antioxidantes/farmacologia , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Compostos de Sulfidrila/farmacologia , Animais , Técnicas Biossensoriais , Células CHO , Cricetulus , Complexo III da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Células HCT116 , Células HEK293 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Suínos , Fatores de Tempo , Transfecção
2.
Biochem Biophys Res Commun ; 439(4): 517-21, 2013 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-24025674

RESUMO

We have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real time intracellular glutathione redox potentials of mammalian cells. This probe enabled detection of media-dependent oxidation of the cytosol triggered by short wavelength excitation. The transient nature of light-induced oxidation was revealed by time-lapse live cell imaging when time intervals of less than 30s were implemented. In contrast, transient ROS generation was not observed with the parental roGFP2 probe without Grx1, which exhibits slower thiol-disulfide exchange. These data demonstrate that the enhanced sensitivity of the Grx1-roGFP2 fusion protein enables the detection of short-lived ROS in living cells. The superior sensitivity of Grx1-roGFP2, however, also enhances responsiveness to environmental cues introducing a greater likelihood of false positive results during image acquisition.


Assuntos
Técnicas Biossensoriais , Glutarredoxinas/química , Luz , Animais , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , Citosol/metabolismo , Dissulfetos/metabolismo , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Oxirredução , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Compostos de Sulfidrila/metabolismo
3.
Exp Biol Med (Maywood) ; 239(4): 394-403, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24586100

RESUMO

The glutathione couple GSH/GSSG is the most abundant cellular redox buffer and is not at equilibrium among intracellular compartments. Perturbation of glutathione poise has been associated with tumorigenesis; however, due to analytical limitations, the underlying mechanisms behind this relationship are poorly understood. In this regard, we have implemented a ratiometric, genetically encoded redox-sensitive green fluorescent protein fused to human glutaredoxin (Grx1-roGFP2) to monitor real-time glutathione redox potentials in the cytosol and mitochondrial matrix of tumorigenic and non-tumorigenic cells. First, we demonstrated that recovery time in both compartments depended upon the length of exposure to oxidative challenge with diamide, a thiol-oxidizing agent. We then monitored changes in glutathione poise in cytosolic and mitochondrial matrices following inhibition of glutathione (GSH) synthesis with L-buthionine sulphoximine (BSO). The mitochondrial matrix showed higher oxidation in the BSO-treated cells indicating distinct compartmental alterations in redox poise. Finally, the contributory role of the p53 protein in supporting cytosolic redox poise was demonstrated. Inactivation of the p53 pathway by expression of a dominant-negative p53 protein sensitized the cytosol to oxidation in BSO-treated tumor cells. As a result, both compartments of PF161-T+p53(DD) cells were equally oxidized ≈20 mV by inhibition of GSH synthesis. Conversely, mitochondrial oxidation was independent of p53 status in GSH-deficient tumor cells. Taken together, these findings indicate different redox requirements for the glutathione thiol/disulfide redox couple within the cytosol and mitochondria of resting cells and reveal distinct regulation of their redox poise in response to inhibition of glutathione biosynthesis.


Assuntos
Citosol/metabolismo , Glutationa/biossíntese , Mitocôndrias/metabolismo , Animais , Butionina Sulfoximina/farmacologia , Células CHO , Linhagem Celular , Cricetulus , Diamida/farmacologia , Inibidores Enzimáticos/farmacologia , Glutarredoxinas/análise , Glutarredoxinas/química , Glutarredoxinas/genética , Dissulfeto de Glutationa/biossíntese , Proteínas de Fluorescência Verde/análise , Células HEK293 , Humanos , Camundongos , Oxirredução , Estresse Oxidativo
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