Identification and characterisation of novel substrates and binding partners of the asparaginyl hydroxylase, FIH

Abstract

The ability of cells to sense and respond to sub-optimal levels of oxygen is a key requirement for organism survival. Many cellular and physiological signalling pathways have been identified as being sensitive to oxygen levels, although remarkably, few of these pathways have been successfully linked with a genuine “oxygen sensing” molecule. Discovery of the 2-oxoglutarate-dependent asparaginyl hydroxylase, Factor Inhibiting HIF (FIH), as an oxygen sensitive regulator of the Hypoxia-inducible Factor (HIF) transcription factors has therefore led to considerable interest in the enzyme as a potential regulator of multiple oxygen-regulated processes. In this work, the known substrate repertoire of FIH was expanded using both yeast 2-hybrid (Y2H) and bioinformatics-based approaches. Potential positives identified in the Y2H screen included a number of proteins which contain an ankyrin repeat structural domain (ARD), and subsequent characterisation of these proteins by in vitro hydroxylation assay suggest that both Fetal Globin Inducing Factor (FGIF) and Serine/threonine-protein phosphatase 6 regulatory ankyrin repeat subunit B (PP6-ARS-B) are both novel substrates of FIH. Concomitant with this discovery, a number of other ARD-containing proteins have been reported as substrates of FIH in the literature, thus suggesting that hydroxylation of ARDs by FIH is common. Comparison of the target sites in these substrates reveals an “FIH preferred sequence” of LXXXXX[-]ΦN, however, it was discovered that FIH can also bind an ARD constrained in its folded state, suggesting that the tertiary fold of ankyrins may also participate in enzyme recruitment. Thus far, hydroxylation of ARDs reported in the literature has not been found to have a significant functional effect on ARD biology. Largely consistent with this, an assessment of the influence of FIH-mediated hydroxylation on IκBα stability and interaction with NFB in this work suggested that the modification has only subtle effects. Furthermore, FIH was found to have no clear effect on the methyltransferase activity of the novel ARD-containing substrate, G9a. In addition to ARD-containing proteins, the Y2H also identified a number of non-ARD-containing proteins which displayed weak interactions with FIH that were inducible by the FIH inhibitor, DMOG. In vitro hydroxylation assays suggest that these proteins are not FIH substrates, and further study will be required to establish the biological significance of these interactions. Overall, this work suggests that FIH interfaces with many partners, and it remains to be determined how these interactions influence the function of FIH, as well as that of its substrates and binding proteins.