Regulation of transcription factors NRF2 and HSF1 in mediating cellular stress responses
The KEAP1/NRF2/ARE pathway is at the forefront of cellular defense. Induction of this pathway is protective against various conditions of stress such as oxidative stress and exposure to electrophiles. Conversely, failure to upregulate the pathway leads to increased disease susceptibility and accelerated pathogenesis. Under basal conditions, transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) is targeted for ubiquitination and proteasomal degradation by a repressor protein Kelch-like (ECH)-associated protein (KEAP1), a substrate adaptor for Cullin 3-based E3 ubiquitin ligase. Inducers of the pathway chemically modify specific cysteines within KEAP1, leading to loss of repressor function, NRF2 accumulation and enhanced transcription of genes encoding a large network of cytoprotective proteins. The cyanoenones are the most potent NRF2 activators known. Cyanoenones bind to KEAP1 covalently and reversibly, and are suitable for chronic in vivo administration. However, the cysteine sensor for these compounds is unknown. Among the cysteine sensors of KEAP1, C151 in the BTB domain and C273 and C288 in the intervening region, are best characterized. In this study using KEAP1-knockout mouse embryonic fibroblasts (MEFs) rescued with wild-type (WT) KEAP1 or cysteine mutants, we tested a series of cyanoenones for their ability to modify specific cysteine sensors in KEAP1. We discovered that remarkably, all compounds of this class specifically target C151 regardless of their molecular shape or size. In addition, primary peritoneal macrophages (PMφ) derived from KEAP1C151S/C151S knock-in mice generated using the CRISPR/Cas9 technology, had higher LPS-induced inflammatory responses than cells from WT animals. Furthermore, at nanomolar concentrations, the tricyclic cyanoenone TBE-31 strongly suppresses LPS-induced inflammatory responses in primary PMφ cells, suggesting the potential for therapeutic use in inflammatory diseases. Another cytoprotective response is the heat shock response (HSR) which is activated when cells are exposed to stressors such as elevated temperatures, heavy metals and cytotoxic compounds. The HSR, mainly mediated by Heat Shock Factor 1 (HSF1), protects the integrity of the cellular proteome through the upregulation of a battery of genes involved in ameliorating proteotoxicity. Several small molecule activators of the KEAP1/NRF2/ARE pathway have been shown to activate HSR through the activation of HSF1. Work in this thesis focused on one such dual activator, the dietary agent phenethyl isothiocyanate (PEITC). We found that PEITC reacts with KEAP1 C151 to stabilize NRF2, and that it is able to activate the HSR by activating HSF1 through the inhibition of heat shock protein 90, the main negative regulator of HSF1. We further showed that PEITC activates p38 mitogen-activated protein kinases (MAPKs). In vitro, all members of the p38 MAPK family rapidly and stoichiometrically catalyze the phosphorylation of HSF1 on S326, a hallmark of HSF1 activation. The use of stable knockdown cell lines and inhibitors indicated that among the p38 MAPK, p38γ is the principal isoform responsible for the phosphorylation of HSF1 at S326 in cells. A protease-mass spectrometry approach confirmed S326 phosphorylation, and unexpectedly, revealed that p38 MAPKs also catalyze the phosphorylation of HSF1 at S303/307, previously known repressive post-translational modifications. Thus, we identified p38 MAPKs as highly efficient catalysts for the phosphorylation of HSF1. Furthermore, our findings suggest that the magnitude and persistence of activation of p38 MAPKs are important determinants of the extent and duration of the HSR. In order to investigate the physiological significance of HSF1 phosphorylation at S326, we used immunofluorescence and western blotting techniques. We found that mitotic cells have high levels of HSF1 pS326 compared to interphase cells. Interestingly, our studies revealed that phosphorylated HSF1 at S326 is concentrated at the midbody of telophase cells, suggesting a potential role for HSF1 during the late-stage of cell division. Further studies are needed to define the role(s) of the HSF1 pS326 during the cell cycle.
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