Substrate specificity of factor inhibiting HIF-1 (FIH-1). (2008)

To detect and respond to the detrimental situation of hypoxia, metazoan cells employ O₂- sensing prolyl and asparaginyl hydroxylases which directly utilise O₂ to hydroxylate and regulate the Hypoxia Inducible transcription Factor-α (HIF-α). This thesis focuses upon the asparaginyl hydroxylase, ‘Factor Inhibiting HIF-1 (FIH-1), which represses HIF-α in normoxia by asparaginyl hydroxylation of its C-terminal trans-Activation Domain (CAD). During hypoxia FIH-1 is inhibited, allowing non-hydroxylated HIF-α to drive expression of over 70 target genes, leading to tissue and cellular changes that increase O₂ supply and reduce its consumption. This response is central to normal physiology and to the pathophysiology of diseases, including stroke and cancer. The pivotal role of FIH-1 in regulating these processes invites its characterisation, as a key cellular O₂-sensor and therapeutic target. This thesis contributes important information by elucidating a novel FIH-1 substrate and by defining numerous FIH-1 substrate recognition determinants. &#13;\nThe first aim was to investigate the cell-fate regulator Notch1 as a potential FIH-1 substrate, due to myriad reports of Notch/hypoxic crosstalk and the discovery by collaborators that FIH- 1 represses Notch1 activity. Mutagenesis, hydroxylation assays, affinity-purification and mass spectrometry techniques enabled definition of two asparaginyl hydroxylations of mouse Notch 1 ankyrin repeat domain (N1945 and N2012), performed by FIH-1 in vitro. These residues were likewise detected to be hydroxylated in mNotch1 expressed in mammalian cells. FIH-1 kinetic assays comparing mNotch1 ankyrin domain with the unstructured hHIF- 1α CAD uncovered major distinctions between substrates; mNotch1 facilitated a 7-fold lower rate of cosubstrate turnover by FIH-1, but affinity was robust (&gt;10-fold higher). Interrogation of the structure/affinity correlate implies FIH-1 binds unstable ankyrins preferentially. Functionally, a non-catalytic mechanism of Notch1 repression by FIH-1 is supported. &#13;\nThe second aim derived from literature analyses implicating threonine and RLL motifs in HIF-α as critical hydroxylation determinants. T796 (hHIF-1α) contacts FIH-1 and is a likely phospho-acceptor, thus a mimetic T796D mutant was generated and its hydroxylation kinetics compared with wildtype hHIF-1α CAD. In vitro, the mutant exhibited a 6-fold greater apparent Km, explaining its constitutive activity in cell-based reporter assays, whereas wildtype hHIF-1α CAD is hydroxylated and thus repressed in normoxia by FIH-1. This indicates that phosphorylation reduces hydroxylation by FIH-1 in vitro and in vivo. &#13;\nThe RLL motif does not contact FIH-1 in vitro however RLL-AAA mutant HIF-α proteins are constitutively active in normoxia, suggesting resilience to hydroxylation within cells. To reconcile these data I predicted that a cellular Factor X functionalises the RLL motif as an FIH-1 binding site. Reporter assays, in vitro kinetic assays and interaction assays +/- lysate confirmed this hypothesis and additionally showed the motif to increase HIF-α protein turnover 8-fold. Numerous mechanisms for Factor X including nuclear export, posttranslational modifications of FIH-1 or HIF-α, and involvement of small molecules, were experimentally examined, but deemed unlikely. Rather, the data imply Factor X to be a proteinaceous facilitator of a HIF-α/FIH-1 complex, thus proteomic capture screens are underway. &#13;\nThis research provides novel insight into FIH-1; its role in Notch/hypoxic crosstalk, its substrate recognition requirements, and its potential functions in cellular O₂-sensing.