The dynamics of redox elements in biologic systems is a major challenge for redox signaling and oxidative stress research. Oxidative stress or signaling events can affect sulfur switches differently, thus creating a variation in the spatial distribution of these redox states, which therefore act simultaneously as regulators and indicators of key cellular functions in both physiological and pathological settings. A gluthatione specific redox-sensitive protein (i.e. a mutant of the Yellow Fluorescent Protein (rxYFP)) has been found to equilibrate in vivo with the gluthatione/gluthatione disulfide (GSH:GSSG) redox couple. rxYFP, employed ratiometrically, allows to generate high resolution maps of the fraction of the reduced protein (R) inside a cell. Here we developed an analytical procedure able to investigate intracellular changes in the glutathione redox-balance, which can occur in live mammalian cells, based on the deconvolution of the histogram of redox maps of 293-TPhoenix human embryonic kidney cells. The intracellular spatial distributions of oxidized and reduced elements have been discriminated. Finally, by transfecting cells with human Glutaredoxin V (GRX-V), an enzyme deputed to maintain reduced the thiol groups of their partner proteins, we can disclose that the significant shift towards more reduced state, with respect to that recovered from non-transfected cells, consists, instead, in a shift towards reduced values of the high R region (reduced), while leaving unaltered the glutathione redox-balance of the intracellular side of the plasma membrane.

Maulucci, G., Pani, G., Labate, V., Mele, M., Panieri, E., Papi, M., Arcovito, G., Galeotti, T., De Spirito, M., Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy., <<BIOSENSORS & BIOELECTRONICS>>, 2009; 25 (Dicembre): 682-687. [doi:10.1016/j.bios.2009.07.038] [http://hdl.handle.net/10807/33061]

Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy.

Maulucci;Giuseppe; Pani;Giovambattista; Labate;Valentina; Mele;Marina; Panieri;Emiliano; Papi;Massimiliano; Arcovito;Giuseppe; Galeotti;Tommaso; De Spirito
2009

Abstract

The dynamics of redox elements in biologic systems is a major challenge for redox signaling and oxidative stress research. Oxidative stress or signaling events can affect sulfur switches differently, thus creating a variation in the spatial distribution of these redox states, which therefore act simultaneously as regulators and indicators of key cellular functions in both physiological and pathological settings. A gluthatione specific redox-sensitive protein (i.e. a mutant of the Yellow Fluorescent Protein (rxYFP)) has been found to equilibrate in vivo with the gluthatione/gluthatione disulfide (GSH:GSSG) redox couple. rxYFP, employed ratiometrically, allows to generate high resolution maps of the fraction of the reduced protein (R) inside a cell. Here we developed an analytical procedure able to investigate intracellular changes in the glutathione redox-balance, which can occur in live mammalian cells, based on the deconvolution of the histogram of redox maps of 293-TPhoenix human embryonic kidney cells. The intracellular spatial distributions of oxidized and reduced elements have been discriminated. Finally, by transfecting cells with human Glutaredoxin V (GRX-V), an enzyme deputed to maintain reduced the thiol groups of their partner proteins, we can disclose that the significant shift towards more reduced state, with respect to that recovered from non-transfected cells, consists, instead, in a shift towards reduced values of the high R region (reduced), while leaving unaltered the glutathione redox-balance of the intracellular side of the plasma membrane.
Inglese
Maulucci, G., Pani, G., Labate, V., Mele, M., Panieri, E., Papi, M., Arcovito, G., Galeotti, T., De Spirito, M., Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy., <<BIOSENSORS & BIOELECTRONICS>>, 2009; 25 (Dicembre): 682-687. [doi:10.1016/j.bios.2009.07.038] [http://hdl.handle.net/10807/33061]
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