Investigating the Role of Cysteamine Dioxygenase in Oxygen Sensing

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This project falls within the "Chemical Biology and Biological Chemistry" EPSRC research area and aims to investigate the oxygen sensing role of cysteamine dioxygenase (ADO). Oxygen homeostasis is essential to various cellular processes within the human body and it is therefore important to understand the body's biochemical response to changes in oxygen levels. ADO has recently been identified as an oxygen-sensitive enzyme involved in protein degradation.1 ADO oxidises the N-terminal cysteine of a variety of proteins, including regulators of G-protein signalling RGS4 and RGS5. Once oxidised the N-terminal cysteine is recognised by arginyl transferases, which add an arginine residue to the N-terminus of the protein. Arginine is destabilising at this position, so degradation of the protein is promoted. This project aims to further investigate this activity of ADO in order to better understand its role in oxygen sensing in humans. The first objective of this project is to identify substrates of ADO in order to understand the scope of its role in oxygen-dependent protein stability. This will involve screening of a variety of polypeptides containing N-terminal cysteines in vitro and analysis of the most active substrates' oxygen sensitivity and other kinetic properties. A consensus sequence will be generated that should aid identification of other possible ADO substrates in the human proteome. This consensus sequence may be further adapted using sequences identified as ADO substrates through an adaptation of an mRNA display selection carried out in collaboration with the Kawamura group in Newcastle. The kinetic data generated will be useful for investigating whether the oxygen sensitivity of ADO catalysis is substrate dependent and therefore the relative importance of the ADO substrates in hypoxia.The second objective is to develop peptide and small molecule inhibitors of ADO, which could be used as orthogonal chemical probes to investigate the biochemical role of the enzyme in cells. Peptides often have more favourable binding properties, while small molecule probes tend to have better physiochemical properties and are able to penetrate cells more easily. Peptide inhibitors will be developed in Oxford using the consensus substrate sequence as well as through an mRNA display selection2 in collaboration with the Kawamura group. Peptide inhibitors may also be used for cocrystallisation studies with ADO by Dr. M. White at the University of Sydney. Finally, it is also proposed to carry out a fragment screen, working with GSK, to find a small molecule metabolite-based inhibitor of ADO. The third objective will be to develop a high-throughput assay capable of detecting the dioxygenated cysteine using recently developed cysteine sulfinic acid probes3. This assay could be used in large-scale screens to select for substrates and inhibitors of ADO, will be invaluable for the fragment screen portion of chemical probe development and will accelerate work towards other objectives. The fourth objective for this project is to carry out mechanistic studies on ADO probing for intermediate species, identifying the rate limiting step and determining the kinetics of oxygen reactivity. The mechanism of cysteine oxidation by thiol dioxygenases is not well understood; information on ADO catalysis may provide a route to better understanding of other thiol dioxygenase mechanisms.