Featured Article: Inhibition of glutathione synthesis distinctly alters mitochondrial and cytosolic redox poise

Abstract

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 + 53DD 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.

Distinct responses of compartmentalized glutathione redox potentials to pharmacologic quinones targeting NQO1

“…The mitochondrial probe exhibits the characteristic tubular shape of the organelle, while in the cytosol fluorescence is diffusely distributed [20]. Ratiometric analysis of time course imaging is expressed through the time resolved changes in excitation ratio of 395 nm and 494 nm channels.…”

“…To target the probe to mitochondria, the mitochondrial matrix targeting sequence adenosine triphosphate synthase protein 9 originating from the fungus Neurospora crassa was used [20]. Grx1-roGFP2 in pQE60 and mito-Grx1-roGFP2 in pLPCX were kind gifts from Dr. Tobias Dick (Cancer Research Center, Heidelberg, Germany).…”

“…The cells were washed twice with Dulbecco’s phosphate- buffered saline supplemented with 5% FBS and 10 mM glucose prior to imaging. Time-lapse images were collected with a fluorescence-enabled inverted microscope (Axiovert 200 M, Carl Zeiss, Feldbach, Switzerland) using an imaging protocol discussed in detail elsewhere [20]. Time-resolved images were acquired every 15 s or 30 s at a 512×512 pixel resolution.…”

“…Images were exported to the Axiovision software in which ROI intensities were calculated and the background fluorescence intensity was subtracted. All results were analyzed by ANOVA, and are expressed as mean ± SD [20]. …”

Methods to study the tumor microenvironment under controlled oxygen conditions

“…To provide more thorough analysis of cell behavior and function under hypoxia, further integration with current biochemical tools is necessary. For example, integrating a microfluidic platform with genetically encoded GFP-based redox sensors could enable measurement of the oxidative state of a cell under hypoxia [8, 62, 63]. Development of methods to quantify secreted molecules would provide insight into cell signaling under hypoxia, however, more research is necessary for current techniques to be adapted under controlled oxygen concentrations [64, 65].…”