Systems-analysis of the Nf-kappaB signalling networks that control levels of reactive oxygen species

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Reactive Oxygen Species (ROS) are oxygen-containing free radicals that are a natural by-product of cellular metabolism. ROS levels also dramatically increase in response to cellular stresses such as heat or UV exposure, tissue wounding, or infection. ROS are highly damaging to cell structures and can result in genetic mutation. Importantly, ROS accumulation is thought to be the cause of ageing, and underlies health problems associated with ageing including impairment of memory and cognitive function. High levels of ROS are also thought to promote the onset of neurodegenerative diseases, diabetes, and cancer. Due to the toxic effects of ROS, cells have evolved a number of genes and biochemical mechanisms in order to prevent the build-up of ROS, and many of these genes are in fact activated as part of the normal stress response. Mutation of these genes can cause the stress response to breakdown, leading to ROS accumulation and subsequent acceleration of the ageing process, and/or the development of diverse pathologies. Through genetic screens, we have found that a gene called Nf-kappaB (or Nf-kB) has a very important role in controlling ROS levels. Nf-kB is a transcription factor that regulates ROS levels by controlling the levels of other genes. Our experiments reveal that loss of Nf-kB activity raises ROS levels, but we also found that excessive Nf-kB activity has the same effect. Therefore, Nf-kB activity must be finely-tuned in order to keep ROS levels low. Using novel genetic screening technology we have developed, we also discovered approximately 200 genes that work together with Nf-kB to control ROS. We predict these genes act as a complex network to modulate the concentration of cellular ROS. While our studies have identified these critical genes, it is currently unknown how the genes work to control ROS levels. Using a number of different technologies and methods, we aim to study the function of these genetic network towards the goal of developing therapeutics that could be used to maintain the proper balance of Nf-kB activity, and thus improve wellbeing during the ageing process. (1) By inhibiting the function of each of these individually we will study how the genes might act to control Nf-kB entry into the nucleus and thus activate transcription. We will also study how inhibition of these genes controls the activity of another important transcription factor called FOXO that we have found regulates metabolism in response to increases in ROS levels. (2) We plan to study how the genes we have discovered are involved in regulating and responding to endoplasmic reticulum (ER) stress and a process called autophagy. Both ER stress and autophagy have been previously linked to the regulation of cellular ROS levels. We will also study how these genes are involved in regulating the metabolic changes in cells that are caused by the exposure of cells to insulin as insulin has also been implicated in controlling ROS levels. (3) Although we identified 213 genes that work together with Nf-kB to regulate ROS levels, there remain many more to be discovered. We will continue to perform further genetic screening in order to determine all the genes that work together with Nf-kB to control the generation of ROS. Many of the genes could novel targets for therapeutics that will improve wellbeing and/or prevent disease. (4) We will develop and implement computational methods in order to map all the relationships that exist between the genes we have discovered. Thus we will generate a map of the Nf-kB gene network that control ROS levels. By developing this map we will be able to determine critical points or 'nodes' in the network that can be targeted by therapeutics to control ROS levels.