Abstract
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.
Highlights
Reactive oxygen species (ROS) are a class of chemical species that promote diverse phenotypes depending on intracellular concentration, localization and cumulative dose over time, spanning the gamut from homeostasis to toxicity [1,2]
The Selective Cancer Killing Hypothesis is based on the idea that some cancers exist at endogenous levels of reactive oxygen species that are higher than healthy cells, so if a patient were systemically treated with a redox-based chemotherapeutic that raises all cells’ levels of reactive oxygen species, only the cancer cells would cross a toxicity threshold
Our model predicts the range of relevant hydrogen peroxide concentrations in the mitochondria of the HeLa model cancer cell line and suggests experimental measurements of tumor cells and tissues that may be useful in quantifying steady state concentrations of this oxidant
Summary
Reactive oxygen species (ROS) are a class of chemical species that promote diverse phenotypes depending on intracellular concentration, localization and cumulative dose over time, spanning the gamut from homeostasis to toxicity [1,2]. Previous work in our group has demonstrated that H2O2 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 H2O2 reaction network. Detailed molecular mechanisms that connect changes in H2O2 with phenotypic responses such as changes in mitochondrial morphology, mitochondrial permeability transition (MPT), and programmed cell death have not been elucidated Since these signaling responses occur during excursions in H2O2 concentration from the basal steady state, we expect that establishing a quantitative range that can be connected with phenotypic responses will help inform whether particular cysteine residues are likely to become directly oxidized [12]. This model represents the first kinetic model of the mitochondrial H2O2 reaction network in a transformed cell line, incorporating the most recent quantitative data specific for HeLa cells
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