Projects

1. Characterisation of novel substrates for the oxygen-sensing asparaginyl hydroxylase FIH-1.

This project is focused on identifying novel substrates in addition to HIF for the oxygen-sensing asparaginyl hydroxylase FIH-1. The majority of enzymes responsible for regulating cellular metabolism have multiple substrates (eg. signal-regulated protein kinases). It is also well documented that cellular responses to hypoxia involve HIF-independent regulation of multiple proteins, which make them probable additional targets of the O2-regulated hydroxylases. The identity of additional hydroxylase targets, and role of such hydroxylation, will shed light on other important cellular processes which are influenced by hypoxia but by poorly understood mechanisms. We have identified a number of additional putative FIH-1 substrates. This project aims to determine whether these proteins are bona fide FIH-1 substrates in vitro and in vivo, and the consequences of hydroxylation on their activity and function. The techniques that will be utilized include protein expression and purification, in vitro hydroxylation assays and mammalian cell-based activity assays. This project is a combination of basic research, but also has the potential to make a significant contribution to our understanding, diagnosis and ultimately even treatment of disease, by identifying and characterizing novel components and mechanisms involved in the cellular response to hypoxia. Further information on the nature of these potential substrates and their characterization are available on request.

2. Investigating the existence and function of a HIF asparaginyl dehydroxylase.

The role of the asparaginyl hydroxylase FIH-1 in hydroxylating the HIF-alpha proteins in an oxygen-dependent manner, and the functional consequences on HIF transcriptional activity have been well characterised. However, FIH-1 is a non-equilibrium enzyme, and as such is only able to catalyse the hydroxylation of HIF-alpha, not the dehydroxylation. The question remains as to whether a cellular dehydroxylase exists, and if so what is its role during hypoxia during development, disease and even high altitude. This project will utilise our knowledge of HIF hydroxylation, the availability of hydroxylated substrate and experience in mass spectrometry (involving a successful collaboration with Prof Jeff Gorman in Brisbane) to answer these questions. The existence of such a dehydroxylase would not only add a level of complexity to our basic understanding of the cellular response to hypoxia and reoxygenation, but may also provide a novel therapeutic target for manipulation of these processes.

Funding Sources

National Health and Medical Research Council of Australia, National Heart Foundation of Australia, Australian Research Council Special Research Centre for the Molecular Genetics of Development.