Mitochondrial H₂O₂ Generation Using a Tunable Chemogenetic Tool To Perturb Redox Homeostasis in Human Cells and Induce Cell Death

Abstract

Among reactive oxygen species (ROS), HO alone acts as a signaling molecule that promotes diverse phenotypes depending on the intracellular concentration. Mitochondria have been suggested as both sources and sinks of cellular HO, and mitochondrial dysfunction has been implicated in diseases such as cancer. A genetically encoded HO generator, d-amino acid oxidase (DAAO), was targeted to the mitochondria of human cells, and its utility in investigating cellular response to a range of HO doses over time was assessed. Organelle-specific peroxiredoxin dimerization and protein S-glutathionylation were measured as indicators of increased HO flux due to the activity of DAAO. Cell death was observed in a concentration- and time-dependent manner, and protein oxidation shifted in localization as the dose increased. This work presents the first systematic study of HO-specific perturbation of mitochondria in human cells, and it reveals a marked sensitivity of this organelle to increases in HO in comparison with prior studies that targeted the cytosol.

Oxidative pentose phosphate pathway and glucose anaplerosis support maintenance of mitochondrial NADPH pool under mitochondrial oxidative stress

“…23,30 In Figure S8). 29,40 The findings shown in Figure 2c are consistent with previous results that a high dose of D-alanine such as 25 mM killed cells through an apoptotic pathway. 29 Next, we tested the functionality of the mitochondrial iNap sensor before implementation of experiments with DAAO system.…”

“…29,40 The findings shown in Figure 2c are consistent with previous results that a high dose of D-alanine such as 25 mM killed cells through an apoptotic pathway. 29 Next, we tested the functionality of the mitochondrial iNap sensor before implementation of experiments with DAAO system. First, we introduced an artificial oxidative stress by stimulating cells with 500 μM diamide, which was previously shown to minimally influence the fluorescence of iNap control sensor.…”

“…28 25 mM for short perturbation times in Hela-DAAO system. 29,41 Similarly, our data demonstrated that 25 mM D-alanine was the threshold concentration that allowed a significant decrease of NADPH pools in mitochondria. Additionally, we assessed whether the absence of carbon source such as glucose or glutamine could influence the mitochondrial NADPH pool during high production of mitochondrial H 2 O 2 .…”

“…The fraction of M + 1 lactate represents the utilization of the PP pathway, whereas that of M + 2 lactate indicates the usage of glycolysis.We hypothesized that flux though the PP pathway could increase when cells were challenged with excessive mitochondrial H 2 O 2 generation rate. When a production rate of H 2 O 2 in mitochondria increased beyond a certain threshold, H 2 O 2 could diffuse out of mitochondria via aquaporin channels and the depolarization of mitochondria has been observed in longer time scales (t > 2 hr), leading to diffusion of H 2 O 2 to the cytosol 16,29. As a result, cytosolic NADPH may decrease, increasing the NADP + level and activating glucose 6-phosphate dehydrogenase 46.…”

Kinetic modeling of H2O2 dynamics in the mitochondria of HeLa cells

“…Previous work in our group has demonstrated that H 2 O 2 perturbations directed to the mitochondrial matrix elicit a marked toxicity in HeLa cells, especially when contrasted against comparable perturbations delivered in the cytosol [10,11]. This toxicity was both concentration-and time-dependent, indicating the importance of a dynamic understanding of the H 2 O 2 reaction network.…”