Kinetic modeling of H2O2 dynamics in the mitochondria of HeLa cells.

Hydrogen peroxide (H2O2) promotes a range of phenotypes depending on its intracellular concentration and dosing kinetics, including cell death. While this qualitative relationship has been well established, the quantitative and mechanistic aspects of H2O2 signaling are still being elucidated. Mitochondria, a putative source of intracellular H2O2, have recently been demonstrated to be particularly vulnerable to localized H2O2 perturbations, eliciting a dramatic cell death response in comparison to similar cytosolic perturbations. We sought to improve our dynamic and mechanistic understanding of the mitochondrial H2O2 reaction network in HeLa cells by creating a kinetic model of this system and using it to explore basal and perturbed conditions. The model uses the most current quantitative proteomic and kinetic data available to predict reaction rates and steady-state concentrations of H2O2 and its reaction partners within individual mitochondria. Time scales ranging from milliseconds to one hour were simulated. We predict that basal, steady-state mitochondrial H2O2 will be in the low nM range (2-4 nM) and will be inversely dependent on the total pool of peroxiredoxin-3 (Prx3). Neglecting efflux of H2O2 to the cytosol, the mitochondrial reaction network is expected to control perturbations well up to H2O2 generation rates ~50 µM/s (0.25 nmol/mg-protein/s), above which point the Prx3 system would be expected to collapse. Comparison of these results with redox Western blots of Prx3 and Prx2 oxidation states demonstrated reasonable trend agreement at short times (≤ 15 min) for a range of experimentally perturbed H2O2 generation rates. At longer times, substantial efflux of H2O2 from the mitochondria to the cytosol was evidenced by peroxiredoxin-2 (Prx2) oxidation, and Prx3 collapse was not observed. A refined model using Monte Carlo parameter sampling was used to explore rates of H2O2 efflux that could reconcile model predictions of Prx3 oxidation states with the experimental observations.

Interpreting Heterogeneity in Response of Cells Expressing a Fluorescent Hydrogen Peroxide Biosensor.

Fluorescent, genetically encoded sensors of hydrogen peroxide have enabled visualization of perturbations to the intracellular level of this signaling molecule with subcellular and temporal resolution. Ratiometric sensors hold the additional promise of meaningful quantification of intracellular hydrogen peroxide levels as a function of time, a longstanding goal in the field of redox signaling. To date, studies that have connected the magnitudes of observed ratios with peroxide concentrations have either examined suspensions of cells or small numbers of adherent cells (∼10). In this work, we examined the response of all cells in several microscopic fields of view to an identical perturbation and observed a striking degree of heterogeneity of fluorescence ratios from individual cells. The expression level of the probe and phase within the cell cycle were each examined as potential contributors to the observed heterogeneity. Higher ratiometric responses correlated with greater expression levels of the probe and phase in the cell cycle were also shown to influence the magnitude of response. To aid in the interpretation of experimental observations, we incorporated the reaction of the reduced probe with peroxide and the reactions of the oxidized probe with glutathione and glutaredoxin into a larger kinetic model of peroxide metabolism. The predictions of the kinetic model suggest possible explanations for the experimental observations. This work highlights the importance of a systems-level approach to understanding the output of genetically encoded sensors that function via redox reactions involving thiol and disulfide groups.

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

Among reactive oxygen species (ROS), H2O2 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 H2O2, and mitochondrial dysfunction has been implicated in diseases such as cancer. A genetically encoded H2O2 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 H2O2 doses over time was assessed. Organelle-specific peroxiredoxin dimerization and protein S-glutathionylation were measured as indicators of increased H2O2 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 H2O2-specific perturbation of mitochondria in human cells, and it reveals a marked sensitivity of this organelle to increases in H2O2 in comparison with prior studies that targeted the cytosol.

Modulating and Measuring Intracellular H2O2 Using Genetically Encoded Tools to Study Its Toxicity to Human Cells.

Reactive oxygen species (ROS) such as H2O2 play paradoxical roles in mammalian physiology. It is hypothesized that low, baseline levels of H2O2 are necessary for growth and differentiation, while increased intracellular H2O2 concentrations are associated with pathological phenotypes and genetic instability, eventually reaching a toxic threshold that causes cell death. However, the quantities of intracellular H2O2 that lead to these different responses remain an unanswered question in the field. To address this question, we used genetically encoded constructs that both generate and quantify H2O2 in a dose-response study of H2O2-mediated toxicity. We found that, rather than a simple concentration-response relationship, a combination of intracellular concentration and the cumulative metric of H2O2 concentration multiplied by time (i.e., the area under the curve) determined the occurrence and level of cell death. Establishing the quantitative relationship between H2O2 and cell toxicity promotes a deeper understanding of the intracellular effects of H2O2 specifically as an individual reactive oxygen species, and it contributes to an understanding of its role in various redox-related diseases.