Disruption of Redox Homeostasis by Enzymatic Activation of a Triakylphosphine Probe in Mitochondria

Abstract

Redox homeostasis is essential for cell function and its disruption is associated with cancer, metabolic and neurodegenerative diseases.Redox balance is largely regulated by the relative concentrations of reduced and oxidized glutathione. In eukaryotes, this ratio is different in each cell compartment, and disruption of the mitochondrial redox balance has been specifically linked to pathologies such as obesity and type II diabetes.Although reduced glutathione can be scavenged using electrophiles, there is a lack of probes that can produce it. In this study, we report an organellespecific reducing agent based on tributylphosphine that selectively reduces oxidized glutathione in mitochondria. This probe is activated by endogenous nitroreductases and subsequently releases tributylphosphine, as well as a fluorescent reporter, within the organelle. Confocal imaging and biological assays in human cells revealed that, counterintuitively, increased reduced glutathione induced oxidative stress through accumulation of superoxide.Transcriptomic analysis was used to establish that mitochondrial redox stress activates a cellular response orchestrated by transcription factor ATF4, which upregulates genes involved in glutathione metabolism.the activity of enzymes that are present only in these organelles to trigger the release of tributylphosphine from a masked precursor. Here, we report the development of such probe, its validation in live human cells, and its application to characterize the cellular response to mitochondrial reductive stress. RESULTS AND DISCUSSIONDesign, Synthesis and Enzymatic Activation. To release tributylphosphine exclusively in mitochondria, we selected endogenous nitroreductases (NTRs) as the activating agent. Although the enzymes that are responsible for this reactivity have not been fully identified in mammalian cells, they are capable of reducing nitroaromatic compounds to amines under normoxic conditions. 12,13 Based on this knowledge, we developed the tributylphosphonium probe 1 with a nitro group as the enzymatically activatable trigger (Scheme 1A). Upon enzymatic reaction, the strong electron-withdrawing nitro group is converted to 1 for Imaging and Detection of Endogenous Superoxide in Live Cells and in Vivo. J. Am. Chem. Soc. 2015, 137, 6837-6843. https://doi.org/10.1021/jacs.5b01881. (18) Lange, P. S.; Chavez, J. C.; Pinto, J. T.; Coppola, G.; Sun, C.-W.; Townes, T. M.; Geschwind, D. H.; Ratan, R. R. ATF4 Is an Oxidative Stress-Inducible, Prodeath Transcription Factor in Neurons in Vitro and in Vivo. J. Exp. Med. 2008, 205, 1227-1242. https://doi.org/10.1084/jem.20071460. (19) Liberman, E. A.; Topaly, V. P.; Tsofina, L. M.; Jasaitis, A. A.; Skulachev, V. P. Mechanism of Coupling of Oxidative Phosphorylation and the Membrane Potential of Mitochondria. Nature 1969, 222, 1076-1078. https://doi.org/10.1038/2221076a0. https://doi.org/10.1093/bioinformatics/btu170. (36) Dobin, A.; Davis, C. A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T. R. STAR: Ultraf...