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Mitochondrial HO generation using a tunable chemogenetic tool to perturb redox homeostasis in human cells and induce cell death Kassi T. Stein, Sun Jin Moon, and Hadley D. Sikes ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00174 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018
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ACS Synthetic Biology
Mitochondrial H2O2 generation using a tunable chemogenetic tool to perturb redox homeostasis in human cells and induce cell death Kassi T. Stein, Sun Jin Moon, Hadley D. Sikes1 Department of Chemical Engineering; Massachusetts Institute of Technology; Cambridge, MA, 02139, USA.
Abstract: 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 concentrationand 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. Keywords: hydrogen peroxide; redox regulation; mitochondria; chemogenetic tools; peroxiredoxin Hydrogen peroxide (H2O2) is a member of a family of molecules known as reactive oxygen species (ROS) and has been demonstrated to induce proliferation, differentiation, and cell death
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. Among ROS, H2O2 is known to behave as a classical signaling molecule,
undergoing specific reactions with cysteine residues to cause various downstream effects 1
To whom correspondence should be addressed (
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has been observed that the intracellular level of H2O2 determines phenotypic outcome, so controlled modulation of H2O2 concentration is desirable in order to probe cellular response 4. Previous studies have investigated cytoplasmic generation of H2O2 and found either no toxic effects6 or only partial toxicity with surprisingly large and lengthy perturbations7. It is thought that subcellular localization of H2O2 generation, in addition to concentration, determines downstream effects1,4. The need for mechanistic studies exploring the effects of targeted, intracellular H2O2 generation has already been identified, as the network of antioxidant reactions confine H2O2 to a small area near its generation site 8–11. This limitation imposed by the interplay of reaction and diffusion rates within the cell highlights the difference between extracellular bolus addition and intracellular peroxide generation
11,12
. Mitochondria are a
compelling organelle for study because they have been suggested as both sources and sinks of intracellular ROS, as well as a hypothesized site for redox signaling 5,13,14. Mitochondria generate superoxide ( O2•− ) at various points in the electron transport chain (ETC) as well as in the Kreb’s cycle, after which it is quickly converted to H2O2 by manganese superoxide dismutase (MnSOD) 5
. The H2O2 levels are managed by the mitochondrial antioxidant network, which is dominated
by the 2-cysteine peroxidase Peroxiredoxin 3 (Prx-3) 5,15. Notably, mitochondrial H2O2 has been implicated in the onset of apoptosis as well as several diseases, such as cancer and diabetes; as such, some therapeutics have targeted mitochondria and mitochondrial ROS
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. However, a dose-response relationship between
mitochondrial H2O2 and cell phenotype, as well as the kinetics involved in this interaction, have not been well established. Synthetic biology tools provide access to this information, enabling elucidation of the strength and activities of compartment-specific networks of antioxidant reactions. In our previous work, we used tunable tools in the cytosol of human epithelial cells to
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ACS Synthetic Biology
establish the concentration- and time-dependence of H2O2-induced cell death 7. We observed only partial toxicity (
