The Spatiotemporal Regulation of the Keap1/Nrf2 pathway

All living organisms have protective systems that allow their successful adaptation and survival under conditions of stress. One major way of protection is the Keap1/Nrf2 pathway. This pathway does not normally operate at its full potential, and can be activated by small molecules (inducers), some of which are present in vegetable-rich diets. Inducers react chemically with cysteine residues of the sensor protein Keap1, leading to stabilisation of transcription factor Nrf2, and consequently to increased expression of a network of genes with protective functions. Previous work has shown that activation of the Keap1/Nrf2 pathway is a very effective strategy for protection against chronic disease, but that in ageing model organisms, the ability to activate Nrf2 is progressively lost. Thus, there is a growing interest in the discovery and development of small-molecule modulators of Nrf2 activity to improve human health and delay ageing.

We have recently developed a system which has allowed us to study the interactions between Nrf2 and Keap1 in single live cells, and discovered that Keap1 uses a unique cyclic mechanism to target Nrf2 for degradation. This project aims to employ our newly-established experimental system in order to: (i) determine the impact of the sensor cysteines of Keap1 which are most commonly modified by electrophiles on the Keap1: Nrf2 complex formation at basal state as well as following inducer treatment; (ii) identify potent non-electrophilic inducers which directly disrupt the protein: protein interaction between Keap1 and Nrf2, and evaluate the ability of such inducers to protect against oxidative stress, a critical contributor to the pathogenesis of essentially all chronic diseases and ageing; (iii) establish the effect on the interaction of Keap1 and Nrf2 of modulating the levels of p21 and p62, two critical protein regulators of the Nrf2 activity which are involved in two fundamental biological processes, apoptosis and autophagy, respectively. Together, these experiments will provide detailed understanding of the mechanisms controlling the interaction between Keap1 and Nrf2. This knowledge is essential for the design and implementation of new strategies aiming the modulation of Nrf2 activity for life-long health and wellbeing.

The transcription factor Nrf2 regulates the expression of more than 1% of the human genome, including detoxification, anti-inflammatory and antioxidant cytoprotective genes. Nrf2 dysregulation is causally-associated with the development of chronic diseases and ageing, and Nrf2 is considered a drug target for disease prevention and healthy ageing. Nrf2 is normally targeted for degradation by its major negative regulator, Keap1. In response to electrophiles and oxidants (termed inducers), which modify specific cysteines within Keap1, Nrf2 is stabilized, accumulates in the nucleus, and activates transcription of cytoprotective genes. Recently, using live cell imaging, we discovered that the Keap1 : Nrf2 interaction follows a cycle whereby the complex sequentially adopts two distinct conformations: "open", in which Nrf2 interacts with one molecule of Keap1, followed by "closed", in which Nrf2 binds to both members of the Keap1 dimer and allows Nrf2 ubiquitination. This project will test the hypothesis that the conformational cycling of the Keap1 : Nrf2 protein complex endows the Keap1/Nrf2-mediated cytoprotective response with regulatory flexibility with respect to both small molecule inducers as well as protein regulators. Specifically, we will determine the effect on the conformational cycling of the Keap1 : Nrf2 complex of: (i) the cysteines of Keap1 which are most commonly modified by electrophiles, (ii) non-electrophilic inducers which directly disrupt the interaction between Keap1 and Nrf2, and (iii) modulating the levels of p21 and p62, two critical protein regulators of Nrf2 activity. Together, these experiments will provide comprehensive understanding of the mechanisms controlling the interaction between Keap1 and Nrf2, leading to new strategies for targeting Nrf2 for life-long health and wellbeing.

The proposed study will have an impact on both academic as well as pharmaceutical research and development. There are already indications that colleagues in industry would probably find aspects of this work of interest. For example, Amgen and AstraZeneca collaborate on an inflammation portfolio, in which Nrf2 activators are being evaluated as potential anti-inflammatory agents. Also, Biogen Idec, which is represented in the UK, has been working on an Nrf2-activating drug, dimethyl fumarate (BG-12), to treat multiple sclerosis. Presumably, Biogen Idec would also be interested in our findings as they will provide detailed insights into the molecular mechanism of action of compounds like BG-12. In addition, the development of new non-electrophilic Nrf2 activators (Aim 2) will be also of great interest to the pharmaceutical industry as they hold the promise of improved safety profile.

Importantly, because some inducers of the Keap1/Nrf2 pathway are present in edible plants, our research benefits scientists and health care professionals who are concerned with improved human nutrition, as well as plant geneticists who are interested in the development of edible plants with enhanced nutritional value. One prominent example is the BBSRC-funded work of Professor Richard Mithen at the John Innes Centre, Institute of Food Research, Norwich Research Park. Professor Mithen has developed the "Beneforté" broccoli, a variety of broccoli with enhanced content of a compound called glucoraphanin, which upon ingestion, is converted to sulforaphane, a potent inducer of the Keap1/Nrf2 pathway. Numerous studies by many research groups, including ours, in various animal model systems have shown that sulforaphane has many health benefits, e.g. it can lower the risk for damage to the retina, heart disease, diabetes, cancer, and is thus considered to be a very promising dietary compound for healthy ageing. Indeed, broccoli or extracts of broccoli have been or currently are in 50 clinical trials for a number of indications, including cardiovascular disease, breast, bladder, lung, prostate and pancreatic cancer, asthma, obstructive lung disease, radiation dermatitis, and even autism (ClinicalTrials.gov).

In addition to academics and scientists, the project will be of interest to the general public. As an RC UK Academic Fellow, Albena Dinkova-Kostova has been involved in public outreach projects. One example is the "Light Up the Lab" project in which Albena presented her work to the 5th and 6th Form pupils in local secondary schools, at the schools themselves during their regular Higher classes, as well as during the course of science exhibitions at the Dundee Science Centre. Albena also has had groups of students spent time in her lab to receive hands-on-experience and appreciation for every day scientific research. All of these activities have been highly successful and have received a very positive feedback from the pupils and their biology teachers. The resources of the Revealing Research team at the University of Dundee to train the post-doctoral assistant in public contact activities are always available to us. As the proposed project progresses, the post-doctoral research assistant will receive training in public engagement and become involved in "Café Science", which provides a regular programme of discussion groups in local public venues such as coffee shops and bookshops. In addition, we will work with the Dundee Science Centre "Café Sensation" to support their series of public lectures about ongoing research in Dundee, which are geared to young people and children. In terms of the wider community, the project will give the staff valuable training and experience in communicating their findings at both scientific and lay levels, which can only have positive benefits to society.