Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2. In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1. Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin ␣. The in vivo and in vitro data indicated that the prothymosin ␣-Keap1 interaction is direct, highly specific, and functionally relevant. Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action. Prothymosin ␣ was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1. In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin ␣ by using prothymosin ␣ overproduction and mRNA interference approaches. Our data attribute to prothymosin ␣ the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin ␣ and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.

While many functions of the p53 tumor suppressor affect mitochondrial processes, the role of altered mitochondrial physiology in a modulation of p53 response remains unclear. As mitochondrial respiration is affected in many pathologic conditions such as hypoxia and intoxications, the impaired electron transport chain could emit additional p53-inducing signals and thereby contribute to tissue damage. Here we show that a shutdown of mitochondrial respiration per se does not trigger p53 response, because inhibitors acting in the proximal and distal segments of the respiratory chain do not activate p53. However, strong p53 response is induced specifically after an inhibition of the mitochondrial cytochrome bc1 (the electron transport chain complex III). The p53 response is triggered by the deficiency in pyrimidines that is developed due to a suppression of the functionally coupled mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). In epithelial carcinoma cells the activation of p53 in response to mitochondrial electron transport chain complex III inhibitors does not require phosphorylation of p53 at Serine 15 or up-regulation of p14 ARF . Instead, our data suggest a contribution of NQO1 and NQO2 in stabilization of p53 in the nuclei. The results establish the deficiency in pyrimidine biosynthesis as the cause of p53 response in the cells with impaired mitochondrial respiration.dihydroorotate dehydrogenase | mitochondrial electron transport chain | NQO1 and NQO2 | p53 tumor suppressor | apoptosis T he mitochondrion is the major power station of the cell that generates most of the cell's supply of ATP. In addition, mitochondria are involved in a range of intracellular processes, such as cell growth and division, differentiation, apoptosis, and intracellular signaling (1). The molecular mechanism of mitochondrial energy transformation involves the electron transport chain (ETC) that consists of electron transfer complexes I-IV embedded in the inner mitochondrial membrane. The ETC converts high energy potential of electrons from NADH and FADH 2 into the energy of electrochemical proton gradient across the inner membrane that drives the synthesis of ATP by the ATP-synthase (complex V). Besides, mitochondria participate in the synthesis of many metabolic intermediates, including the de novo biosynthesis of pyrimidines. The latter process is catalyzed by dihydroorotate dehydrogenase (DHODH), an FMN flavoprotein in the inner mitochondrial membrane, which transfers electrons from dihydroorotate to ubiquinone of the ETC for further oxidation (2).The p53 tumor suppressor mediates important quality control functions by limiting proliferation and survival of abnormal or damaged cells. Mitochondria are critically important for the p53-mediated cell death, because p53 controls transcription of several genes that affect the release of mitochondrial cytochrome c (3). In addition, p53 can induce a transcription-independent apoptosis through the direct interaction with Bcl-2 family proteins (4). On the ...

A HeLa cell line expressing the green fluorescent protein fused to the SV40 T-antigen nuclear localization signal (EGFP-NLS) was established. Fluorescence in these cells was confined to the nuclei. After poliovirus infection, cytoplasmic fluorescence in a proportion of cells could be detected by 1 h postinfection (p.i.) and in virtually all of the fluorescent cells by 2 h p.i. The relocation could be prevented by cycloheximide but not by inhibition of poliovirus replication by guanidine. HCl. Nuclear exit of a protein composed of three copies of GFP fused to the NLS also occurred upon poliovirus infection. A similar redistribution of EGFP-NLS took place upon infection with coxsakievirus B3 and, to a lesser extent, with vesicular stomatitis virus. The EGFP-NLS efflux was not due to the loss of NLS. Thus, some positive-strand and negative-strand RNA viruses trigger a rapid nonspecific relocation of nuclear proteins.

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