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Mitochondria are required for ATM activation by extranuclear oxidative stress in cultured human hepatoblastoma cell line Hep G2 cells.

Significance Statement

Aerobic metabolism is the most efficient way to synthesize high-energy compounds such as ATP, but it acts as a two-edged sword by leading to the formation of reactive oxygen species (ROS). In order to overcome the stressful, risky circumstances, aerobic organisms have evolved defenses against the toxic effects of oxygen. Ataxia-telangiectasia mutated is thought to be involved in the antioxidative mechanisms by its oxidative activation through the formation of disulfide cross-links within the homodimer, and the Ataxia-telangiectasia mutated activation can occur in the absence of DNA damage response (DDR). The ROS-sensing cysteine residue (C2991) is conserved in terrestrial vertebrates but not in marine animals, suggesting that the evolved ATM might serve to protect these vertebrates from oxidative stress. We think that it is reasonable to assume that the DDR-independent activation of ATM occurs at extranuclear sites. The objective of this study was to clarify the specific location where Ataxia-telangiectasia mutated is activated by oxidative stress, using a hepatoblastoma cell line Hep G2 cells. At first, we determined a concentration of H2O2 that activated Ataxia-telangiectasia mutated in the absence of DDR, and the localization of Ataxia-telangiectasia mutated was investigated using two subcellular fractionation methods. As a result, each mitochondria-containing fraction by both methods was enriched in ATM and active Ataxia-telangiectasia mutated, suggesting that the DDR-independent, oxidative activation of Ataxia-telangiectasia mutated occurs in mitochondria. Further study using Rho 0-Hep G2 cells revealed that mitochondria are required for the oxidative activation of Ataxia-telangiectasia mutated. These findings strongly suggest that, in response to oxidative stress, Ataxia-telangiectasia mutated can be activated in mitochondria in a DDR-independent manner.

Journal Reference

Morita A, Tanimoto K, Murakami T, Morinaga T, Hosoi Y.

Biochem Biophys Res Commun. 2014 Jan 24;443(4):1286-90.

Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan; Department of Radiological Science, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8509, Japan. Electronic address: [email protected] and

Department of Radiation Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan; Department of Radiation Biology, Graduate School of Medicine, Tohoku University, Sendai 980-8575, Japan. Electronic address: [email protected]

Abstract

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in DNA damage response (DDR). A recent study reported that oxidized Ataxia-telangiectasia mutated can be active in the absence of DDR. However, the issue of where ATM is activated by oxidative stress remains unclear. Regarding the localization of Ataxia-telangiectasia mutated, two possible locations, namely, mitochondria and peroxisomes are possible. We report herein that Ataxia-telangiectasia mutated can be activated when exposed to hydrogen peroxide without inducing nuclear DDR in Hep G2 cells, and the oxidized cells could be subjected to subcellular fractionation. The first detergent-based fractionation experiment revealed that active, phosphorylated Ataxia-telangiectasia mutated was located in the second fraction, which also contained both mitochondria and peroxisomes. An alternative fractionation method involving homogenization and differential centrifugation, which permits the light membrane fraction containing peroxisomes to be produced, but not mitochondria, revealed that the light membrane fraction contained only traces of ATM. In contrast, the heavy membrane fraction, which mainly contained mitochondrial components, was enriched in ATM and activeATM, suggesting that the oxidative activation of ATM occurs in mitochondria and not in peroxisomes. In Rho 0-Hep G2 cells, which lack mitochondrial DNA and functional mitochondria, ATM failed to respond to hydrogen peroxide, indicating that mitochondria are required for the oxidative activation  of ATM. These findings strongly suggest that ATM can be activated in response to oxidative stress in mitochondria  and that this occurs in a DDR-independent manner.

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