Abstract
Oxidative stress has been associated with numerous degenerative diseases and disorders, as well as cancer and the process of ageing. In higher animals, the response to oxidative stress is largely regulated by the master transcription factor Nrf2, which controls the transcription of cytoprotective genes, and its inhibitor Keap1 which functions as a “sensor” of oxidative stress. Keap1-Nrf2 pathway is known to be conserved across vertebrates and certain invertebrates, but its evolution is yet to be described and it is currently unknown if microbes such as yeasts and bacteria possess this pathway. This thesis examines microbial genomes for evidence of Keap1-Nrf2 pathway, investigates the evolution of this pathway over geological time, and assesses the potential for activation of Nrf2-controlled cytoprotection by microbially produced small compounds.The novel software for identification of distant homologs was developed and utilized to study the homologs of Keap1 and Nrf2 proteins in genomes of animals and microorganisms. The evolution of Keap1-Nrf2 pathway was reconstructed by phylogenetic studies, and the time-frame of evolution was calibrated using the fossil record. The existence of Keap1-Nrf2 pathway in fungi was also examined empirically by utilizing high-throughput proteomics to quantify the stress response mechanisms of an UV-tolerant yeast model. Structure based virtual screening was employed to identify microbial natural products with potential to activate human Nrf2 pathway by inhibiting the Keap1-Nrf2 binding, and the prospective in-silico activators of Nrf2 were tested in vitro by fluorescence polarisation and thermal shift assays to detect competitive inhibition of human Keap1-Nrf2 binding.
In-silico analyses identified that the Keap1-Nrf2 pathway exists in all major eukaryotic phyla, ranging from fungi to mammals, and that Nrf2 evolved under a selective pressure incurred by the rise of oxygen levels over geological time. The in-silico virtual screen identified the potential for competitive inhibition of Keap1-Nrf2 binding by mycosporine-like amino acids (MAAs), small compound UV-protective and antioxidant metabolites of marine microorganisms. This activity of MAAs was tested empirically, and the MAAs shinorine and porphyra-334 were confirmed to competitively inhibit the human Keap1-Nrf2 interaction in vitro. The results presented herein indicate that natural products of microorganisms, such as MAAs, are the prospective compound leads for the design of novel therapeutics to target activation of the human Keap1-Nrf2 pathway for treating degenerative diseases of oxidative stress, whilst avoiding the off-target effects of currently utilized Nrf2 activators.
| Date of Award | 2017 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Paul Long (Supervisor), David Barlow (Supervisor) & David Thurston (Supervisor) |