Role of AMPK for the Nrf2-dependent celluar stress response

The proposed project deals with the question how the supply with energy determines to what extent a cell is able to resist stress (on a molecular level). We will focus on two proteins (i) AMP-activated kinase (AMPK) and (ii) nuclear factor 2 related factor 2 (Nrf2). The first is a crucial sensor of the supply with nutrients and is activated when energy gets low. Activated AMPK enhances processes that provide energy (e.g. respiratory catabolism) and inhibits energy consuming processes (e.g. cholesterol biosynthesis). The second is a transcription factor that is usually activated when the cell is hit by a harmful insult, such as oxygen radicals or toxins. Upon activation Nrf2 initiates transcription of genes which help the cell to detoxify and get rid of damage, making Nrf2 an important protein to remain untouched by environmental and endogenous stress. Our previous studies demonstrated that a specific Nrf2 target gene, i.e. heme oxygenase 1 (HO-1), is stronger expressed when AMPK is activated, suggesting a positive cooperation between AMPK and Nrf2. The current project wants to decipher how AMPK brings about the boost of the Nrf2/HO-1 axis on a molecular level. We will investigate whether AMPK has an influence on the cellular localization of Nrf2, whether AMPK alters binding strength or duration of Nrf2 to its target DNA, whether AMPK has an impact on the assembly of nuclear Nrf2 transactivation complex and/or whether AMPK alters the Nrf2 protein by leading to attachment of posttranslational modifications. We will further have a look if AMPK influences all Nrf2 target genes or only a subset of the 200 known genes under the control of Nrf2. After performing these basic studies in mouse embryonic fibroblasts obtained results are to be confirmed in more sophisticated cell models and in vivo. In order to address all these issues we will use an extensive set of methods, including cell culture, expression analyses, protein-protein/DNA interaction studies, mass spectrometric analyses as well as live in vivo imaging (micro-computer tomography and bioluminescence imaging). The project will provide important and novel insight into the incompletely understood crosstalk between cellular energy homeostasis and stress signalling. The findings may foster the understanding why and how caloric restriction (AMPK activation) on a an organismal level accounts for the observed life span prolongation due to increased stress resistance and ultimately offer new inspiration for the development of drugs (dual Nrf2 and AMPK activators) aiming for healthy aging.