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Title: Studies on FIH interactions
Author: Leissing, Thomas
ISNI:       0000 0004 7430 5667
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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The ability to adapt to variations in oxygen levels is essential for higher organisms. In animals, the chronic response to hypoxia (limiting oxygen availability) is substantially regulated by the hypoxia inducible factors (HIFs), which are αβ-heterodimeric transcription factors. The activity and stability of HIF-α subunits are regulated by 2-oxoglutarate (2OG) dependent oxygenases. When sufficient oxygen is present (normoxia), prolyl-4-hydroxylation by the prolyl hydroxylase domain containing proteins (PHDs) signals for HIF-α degradation by promoting binding to the von Hippel-Lindau (VHL) tumour suppressor protein, an E3 ubiquitin ligase. In hypoxia, PHD activity slows and the HIF-α subunits are stabilised leading to increases in HIF-α levels, heterodimerisation with HIF-β, and consequent increased transcription of HIF target genes (e.g. erythropoietin, EPO). Factor Inhibiting HIF (FIH), is an oxygenase that, at least in higher animals, regulates HIF-1/2α activity by catalysing C-3 hydroxylation of a single asparagine in the HIF-α transcriptional activation domain (CAD), thereby blocking the binding of HIF to the CBP/p300 transcriptional coactivators. Pharmaceutical companies have developed PHD inhibitors to increase endogenous EPO levels via HIF-α stabilisation, that are in late stage clinical trials for anaemia treatment in chronic kidney disease. Using FIH as a PHD surrogate, the structural basis for PHD inhibition by clinical candidates was studied. Together with solution studies, the crystallographic results reveal PHD inhibition occurs via 2OG competition as well as by varying degrees of substrate competition. In addition to HIF-α, FIH interacts with multiple ankyrin repeat domain (ARD)-containing proteins, many, but not all, of which are hydroxylated by FIH. The physiological roles of FIH-mediated ankyrin hydroxylation are not fully understood, but ARD proteins are able to compete with HIF for FIH activity and hence may contribute to the regulation of the hypoxic response. To screen for small molecules that could potentially lead to a substrate specific inhibitor, blocking FIH-ARD interactions, thus promoting FIH mediated suppression of HIF transcriptional activity, a crystallographic fragment screen was established and performed for FIH; several small molecules binding to the substrate binding site of FIH were identified. FIH-ARD interactions were studied for the ARDs of Transient Receptor Potential (TRP) ion channels. Five crystal structures of FIH in complex with TRP channel derived peptides were obtained; analyses of their structures reveal differential modes of FIH substrate binding. Attempts were made to obtain structures of FIH-ARD protein-protein complexes included re-design of the FIH crystallisation construct and disulfide cross-linking approaches. Evidence was accrued for a novel FIH ARD hydroxylation, i.e. the double hydroxylation of two asparagine residues within a single ARD, in the case of Apoptosis Stimulating Proteins of p53 (ASPP) and with related ARD proteins. Potential effects of FIH-mediated hydroxylation on ASPP2 protein function were investigated using interaction studies in cells. To better understand the mechanisms of 2OG oxygenases, methodologies for X-ray Electron Free Lasers (XFEL) measurement of 2OG oxygenases and related enzymes were developed and the first XFEL room-temperature structures for 2OG oxygenases were achieved. Overall, the results extend the scope of FIH catalysis, provide new insights into hydroxylase inhibition, and identify XFEL as a useful technique for studying 2OG oxygenase mechanisms.
Supervisor: Schofield, Christopher ; Lu, Xin Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available