Investigating the role of cysteamine dioxygenase in oxygen sensing

Many organisms have developed systems that allow them to sense and respond to changes in oxygen levels, and so maintain oxygen homeostasis. Recently, a new oxygen-sensing system was identified in animals, which involves a thiol dioxygenase (TDO) enzyme, cysteamine dioxygenase (2-aminoethanethiol dioxygenase or ADO). In normoxia, ADO catalyses the oxidation of N-terminal cysteine (Nt-Cys) on target proteins to cysteine sulfinic acid, which triggers protein degradation via the Arg/N degron pathway. ADO activity is inhibited by hypoxia, so its target proteins are stabilised. In this manner, ADO confers oxygen-dependent stability on its protein substrates, similar to the action of the related plant cysteine oxidases (PCOs).
Work conducted in this Thesis aimed to increase understanding of ADO and its role in the hypoxic response. Only three protein substrates of ADO were identified in the initial study (regulators of G-protein signalling RGS4/5 and a chemokine IL32). In this Thesis, broad in vitro screening of ADO activity towards additional Nt Cys-bearing sequences revealed that it is highly substrate specific, preferentially catalysing oxidation of peptides with basic or aromatic residues after the Nt-Cys. Interestingly, this preference was also observed when ADO was tested with PCO substrates, although catalytic activity towards these was significantly lower than towards ADO’s known human substrates.
ADO activity towards peptides proved to be broadly a good indicator of ADO activity as manifest in cells with N-terminal substrate sequence fusion protein reporter assays. However, two sequences appeared to not be ADO substrates in this cell assay, contrary to in vitro data with corresponding peptides. Furthermore, inconsistencies with cellular assays examining ADO activity towards full-length putative substrates highlighted that other factors, perhaps including protein folding, are important to consider when using in vitro data to predict in cellula regulation by ADO. No novel physiological protein substrates were identified in this Thesis. However, the results presented greatly increase understanding of ADO activity and will be useful for prediction of novel substrates in the future. The unexpected in cellula results noted above (i.e., the lack of regulation in cellula of some sequences despite positive in vitro data) led to investigation of Nt Cys acetylation and its influence on ADO activity, through a collaboration with Thomas Arnesen and colleagues at the University of Bergen. Nt Cys acetylation was, for the first time, demonstrated to be catalysed by N terminal acetyltransferase A (NatA). Notably, NatA activity was also shown to be substrate dependent, with a preference contrasting to that of ADO. NatA preferentially catalyses acetylation of peptides with acidic or polar residues after the Nt-Cys. This suggests that ADO and NatA have evolved to modify distinct pools of Nt Cys proteins. Nt Cys acetylation was also shown to abrogate ADO activity (and vice versa for oxidation and NatA activity), raising the possibility that some Nt Cys sequences could be shielded from ADO-dependent degradation by co-translational acetylation.
Furthermore, peptide and small molecules were characterised as ADO inhibitors, with substrate-based Nt selenocysteine peptides identified as the best candidates for specific and potent chemical probes. Finally, investigation of ADO’s structure using an in silico model and published crystal structures identified features within the active site that may contribute to substrate binding and explain some of the experimental observations made in this Thesis.
Overall, the results presented in this Thesis contribute to understanding of ADO’s role in oxygen-dependent protein signalling, including the extent and determinants of its protein substrates, its interaction with other N terminal modifications, and its structure-function relationship. Knowledge of ADO’s activity on novel protein substrates, and inhibitors thereof, can help to characterise the hypoxic response and could be important in disease conditions, such as ischaemia and cancer.

Keywords: N-degron pathways
N-terminal modifications
oxygen sensing
thiol dioxygenases
Advisor: Flashman, E
Ratcliffe, P
Bush, J