Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo

Abstract

Summary Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo , without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo .

Respiratory chain signalling is essential for adaptive remodelling following cardiac ischaemia

“…Mitochondria were isolated from cardiac tissue as described previously . Mitochondrial oxygen consumption and ROS production were measured using an Oroboros O2k oxygraph (Oroboros Instruments) at 37°C.…”

“…Mitochondria were isolated from cardiac tissue as described previously. 43 Mitochondrial oxygen consumption and ROS production were measured using an Oroboros O2k oxygraph (Oroboros Instruments) at 37°C. Standard experimental set-up was 200 µg of mitochondrial protein per mL of mitochondrial respiration buffer (120 mmol/L KCl, 1 mmol/L EGTA, 10 mmol/L HEPES, 5 mmol/L KH 2 PO 4 , pH 7.2; Sigma, P5405, E3889, H3375, P5655) along with 30 μL of 1 mg/mL superoxide dismutase (Sigma, S5395), 30 µL of 1 mg/mL horseradish peroxidase (Sigma, 77332), 30 µL of 10 mg/mL fatty acid-free-BSA (Sigma, A6003) and 5 µL of Amplex Red Reagent (ThermoFisher, A12222) for hydrogen peroxide detection.…”

Cytoplasmic and Mitochondrial NADPH-Coupled Redox Systems in the Regulation of Aging

“…In addition in rat liver 59% of the total cellular NADP(H) was reported to be present in mitochondria [58], although mitochondria only make up 10–15% of the cell volume. Therefore it may take many hours to days for cells to completely recover from oxidation of GSH if the cells are able to recover at all [64].…”

Auranofin Mediates Mitochondrial Dysregulation and Inflammatory Cell Death in Human Retinal Pigment Epithelial Cells: Implications of Retinal Neurodegenerative Diseases

“…Trx1 and its reductase TrxR1 are present in the cytosol and nucleus while Trx2/TrxR2 redox system is in the mitochondrion. Therefore, Trx/TrxR redox system plays an important role in scavenging reactive oxygen species (ROS) and maintaining reduced state of proteins in their functionally active sites and cell survival (Devi et al, 2017; Stephenson et al, 2018; Booty et al, 2019). Thus, TXNIP inhibition of the Trx/TrxR system causes oxidative stress, mitochondrial dysfunction, and retinal cell death in diabetes (Perrone et al, 2010; Singh and Perrone, 2013).…”